Embryology - User contributions [en-gb]

User:Z3417753

2014-10-29T00:53:42Z

Z3417753: /* Lab Attendance */

{{StudentPage2014}}

==Lab Attendance==
* Lab 1 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 12:54, 6 August 2014 (EST)
* Lab 2 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:21, 13 August 2014 (EST)
* Lab 3 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 20 August 2014 (EST)
* Lab 4 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:45, 27 August 2014 (EST)
* Lab 5 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:48, 3 September 2014 (EST)
* Lab 6 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:50, 10 September 2014 (EST)
* Lab 7 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:31, 17 September 2014 (EST)
* Lab 8 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 24 September 2014 (EST)
* Lab 9 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:27, 8 October 2014 (EST)
* Lab 10 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:11, 15 October 2014 (EST)
* Lab 11 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:26, 22 October 2014 (EST)
* Lab 12 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:53, 29 October 2014 (EST)

==Online Assessment 1==

===Article 1===
<pubmed>23148203</pubmed>

This study is an analysis of the optimal time from oocyte to preimplantation embryo development for biopsy and preimplantation genetic screening. The discovery of the optimal time can then be used to detect any abnormal chromosomal separation patterns in embryos from older mothers (>40 years old). The study was a longitudinal cohort study involving 9 infertile couples and 21 sets of complete chromosomal screening data, including polar bodies 1 + 2 and their corresponding blastomeres and trophectoderm samples.

METHODS →infertile couples with a good response to controlled ovarian stimulation were enrolled in the study and underwent IVF. Polar bodies, blastomeres and trophectoderm samples were biopsied and analysed by array comparative genomic hybridisation. The chromosomal segregation patterns were analysed from these results and used to deduce the origin of aneuploidy. The results were also used to examine the accuracy of polar body and cleavage-stage preimplantation genetic screening strategies.

RESULTS → Since preimplantation genetic screening tests have been conducted at different times throughout the preimplantation window, it is possible that critical bits of information regarding chromosomal segregation patterns have been missed. Thus, by performing such tests at an optimal time, we are better able to understand these meiotic chromosomal segregation patterns and therefore potentially increase the success rates of in-vitro fertilisation. This study uses a sequential chromosome analysis of polar bodies and their corresponding embryos at both the cleavage and blastocyst stages in order to work out what stage is best to perform these genetic screening tests and biopsies, potentially increasing IVF success rate. The study showed that testing at the polar body stage was least accurate due to the high incidence of post-zygotic events and discovered that performing these tests later on in development (at the blastocyst stage) may produce more reliable results for the screenings, thereby achieving better chromosomal segregation pattern data. These results can now go on to be used for IVF research.

===Article 2===

<pubmed>23477909</pubmed>

This study examines the accuracy of using array comparative genomic hybridisation (array CGH) techniques for the analysis of first and second polar bodies in predicting aneuploidies of maternal meiotic origin in the cleavage stage embryos of women of advanced maternal age. It is known that aneuploidy is a common cause of pregnancy failure, miscarriage and abnormal pregnancy and most aneuploidy is due to maternal meiotic origin and increases exponentially as the mother approaches menopause.

METHOD → 20 couples requesting preimplantation genetic screening for advanced maternal age (=greater than or equal to 35 years old) and repeated implantation failure (more than 3 cycles), previous aneuploidy pregnancy or recurrent first trimester miscarriage underwent 16 controlled ovarian hyperstimulation cycles and 7 natural fresh cycles. Male partners had sperm parameters within the normal range except for 2 which had oligoasthenoteratozoospermia. Oocytes were retrieved by ultrasound-guided transvaginal aspiration 36 hours after beta-hCG administration. Once the oocytes were retrieved, biopsy of the first polar body was performed and the oocyte was inseminated using intracytoplasmic sperm injection. The following morning, each oocyte was checked for prouclei and extrusion of the second polar body to confirm fertilisation. The second polar body was then biopsied. The polar bodies were then analysed using array CGH analysis and the zona pellucida layer of the oocyte was dissolved. The zona-free embryo then underwent whole genome amplification and array CGH analysis in the cleavage stage.

RESULTS → It has been demonstrated in previous studies that a high correlation exists between the chromosomal status presented from polar body analysis and the actual chromosomes present in the zygotes of older mothers. Due to these results, this study uses polar body analysis and array CGH analysis of mature fertilised oocytes, to identify errors in meiosis within the polar bodies as well as the corresponding cleavage stage embryos. The results of the current study showed that nearly ALL aneuploidies detected in cleavage stage embryos were associated with copy number changes in the polar bodies (93%), indicating the high capability of polar bodies being used to predict aneuploidy and what is actually happening within the embryo.

--[[User:Z8600021|Mark Hill]] These are good articles and summaries. Reference link is formatted correctly (5/5)

==Online Assessment 2==
[[File:Fusion of two pairs of blastomeres inside 4-cell embryos.png|800px]]

Fusion of two pairs of blastomeres inside 4-cell embryos<ref><pubmed>23227157</pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515590/figure/pone-0050029-g003/]</ref>

<references/>

--[[User:Z8600021|Mark Hill]] Image appropriate for assessment and all associated information formatted correctly. You may want to include species (mouse) information with the image. (5/5)

==Online Assessment 3==
===Current Research Models and Findings===
--[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 22:57, 26 August 2014 (EST)

<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>

--[[User:Z8600021|Mark Hill]] These are relevant references, a slingle line describing why you have selected these would have been also useful to include. (4/5)

==Online Assessment 4==
===Cord Stem Cell Article Findings===

<pubmed>25101638</pubmed>

This article identifies acute liver failure as a devastating and debilitating illness that occurs within a short period of time, ultimately resulting in death of the patient if proper treatment is unavailable or it is simply too late to treat. It further identifies liver transplantation as the most effective treatment to date however, its application is limited due to an elevated risk of organ rejection and lack of liver donors. It is also known that human umbilical mesenchymal stem cells (hUCMSC) have the potential to differentiate into hepatocyte-like cells, functioning very similarly to hepatocytes as well as secrete certain factors to stimulate the proliferation of nearby hepatocytes, thereby promoting the rejuvenation of the host liver cells. The author hypothesised that by decreasing the amount of manipulation received by the mesenchymal stem cells in vitro, the carcinogenic risk was reduced. As a result, the therapeutic effect (amount of liver repair) of concurrently acting hUCMSC’s and hepatocyte-like cells can be ascertained by studying and comparing the two synchronous actions in acute liver failure mouse models.

The study induced acute liver failure in mouse models using D-galactosamine and lipopolysaccharide, causing the death of approximately 50% of the mice (necrosis of more than 50% of the hepatocytes). The mouse models’ therapeutic effects were then compared before and after the mesenchymal stem cells were differentiated into hepatocyte-like cells, by transplanting and injecting the cells into the tail vein. The results showed that almost ALL mouse were saved by the injection of the hepatocyte-like cells. Similarly, the injection of the hUCMSC’s also demonstrated their capability to repair liver damage, however, the population of these cells tested via the expression/ presence of human hepatocyte growth factor was minimal, suggesting that they allow the reversal of acute liver failure by differentiating into hepatocyte-like cells.

Overall, these results suggest that hUCMSC’s and hepatocyte-like cells are just as effective in therapeutic treatment of acute liver failure in mouse models and that hUCMSC’s play a larger role in stimulating the host hepatocyte repair.

===Vascular Shunts===

Three major vascular shunts exist within the circulatory system of the foetus:

1. FORAMEN OVALE --> the opening in the interatrial septum (wall between left and right atrium) that allows the flow of blood from the right atrium to the left atrium and has a valve to prevent backflow during the fetal period. It soon closes once right atrial pressure increases.
The foramen ovalis then becomes the FOSSA OVALIS postnatally.

2. DUCTUS ARTERIOSUS --> muscular vessel that connects the pulmonary trunk to the aorta, thereby diverting bloodflow to the lungs and going straight into the aorta. After birth, as the amount of oxygen increases, the smooth muscle in the walls constricts closing off the passage.
As the ductus arteriosus degenerates, all that is left behind if the LIGAMENTUM ARTERIOSUM.

3. DUCTUS VENOSUS --> a blood vessel that branches from the umbilical vein, allowing oxygenated blood from the placenta to be diverted from the fetal liver to the fetal heart.
This shunt closes slowly during infancy and degenerates into the LIGAMENTUM VENOSUM.

==Online Assessment 5==
Laryngeal-tracheo-oesophageal Cleft is a rare congenital anomaly where there is an abnormal posterior communication between the larynx and pharynx, extending down between the trachea and oesophagus.

Normally, the larynx develops simultaneously from the endoderm (arising from the foregut region) and the mesenchyme (arising from the 4th + 6th pharyngeal arches. The division of the foregut is due to the fusion of the lateral walls of the foregut in the region of the larynx, thereby forming a septum that divides the foregut into a central part = LARYNGEAL-TRACHEAL TUBE as well as a dorsal portion = OESOPHAGUS. The mesenchymal portion (= TRACHEAL-OESOPHAGEAL SEPTUM) is located between the digestive and respiratory tracts and is the result of the separation of the two tracts. Apoptotic epithelial cells are also present at this septum, mainly in the ventral portion, but inactive in the dorsal portion.

There are a few models that explain tracheal-oesophageal anomalies, including Laryngeal-tracheo-oesophageal Cleft:

• INTRAEMBRYONIC PRESSURE → an intense curvature of the cervical region during heart development places pressure upon the oesophagus and as a result displaces it, leading to growth abnormalities.

• EPITHELIAL OCCLUSION → the oesophagus is solid during a stage of development but it is soon recanalised. If the recanalisation does not occur, growth abnormalities may occur.

• VASCULAR OCCLUSION → an abnormally communicating vessel could lead to avascularisation in the foregut, resulting in abnormalities. In the case of Laryngeal-tracheo-oesophageal Cleft, this means the laryngeal region.

• DIFFERENTIAL CELL GROWTH → abnormal cell growth in the ventral or dorsal part of the developing trachea or oesophagus could result in defects of the two tracts.<ref><pubmed>22151899</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22151899]</ref>

<references/>

==Online Assessment 6==

===Research Article on Development of the Pancreas===
<pubmed>24375815</pubmed>

This article explores the role of pancreas-specific transcription factor 1a (PTF1a) in development of the pancreas, but makes a note of the difficulty associated with obtaining embryonic tissue specimens for experimentation. Due to the limitations associated with such embryonic material, embryonic stem cells (ESCs), which can be differentiated in vitro are used as a model system to study and examine the role of PTF1a in the development of the pancreas.
The study uses cell cultures, quantitative PCR, immunofluorescent staining, flow cytometry and western blot staining to demonstrate that PTF1a is required very early in development for arrangement of the pancreas from the foregut endoderm. The study shows that PTF1a drives differentiation of pancreatic cells from embryonic stem cells and this determines PTF1a to be an initiator of pancreatic differentiation in the form of ductal, endocrine and exocrine cells. The ectopic expression of PTF1a stimulated the ESCs to start differentiating into pancreas, causing the cells to activate PDX1 expression in bud-like structures that looked like early pancreas in vivo. The study also found that retinoic acid and nicotinamide signaling could regulate the ratio of endocrine to exocrine cell differentiation.
The future implications of this study may involve further studies utilizing the notion of the importance of activating PTF1a in the development of enhanced pancreatic differentiation for creating ESC-derived insulin (beta cells) expressing cells.

===Embryonic Layers and Tissues that Contribute to Tooth Development===

Teeth are part of the integumentary system and are formed by epithelial as well as mesenchymal interactions during development. They are largely formed by ECTODERM of the first pharyngeal arch, MESODERM and receive a major contribution from NEURAL CREST ECTOMESENCHYMAL cells. The neural crest mesenchymal cells change due to the enamel epithelium.

==Online Assessment 7==

'''TIME COURSE OF EMBRYONIC DEVELOPMENT OF THE HUMAN TESTIS'''

During fertilisation, genes determine the sexual fate of the organism and whether the organism is male or female is only revealed in fetal development when development of the external genitalia finally occurs. The presence of the Y chromosome leads to the development of testes in humans and their development is dependent on a single gene located on this Y chromosome known as Testis-determining factor (TDF).

When observing the morphology and cell biology of the developing testis, it is important to note that much of the research conducted on the subject involved the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy.

The next stage in embryonic development of the human testis is the formation of the genital ridge in an initial phase. This genital ridge is representative of the ambisexual stage in human embryos and is a bipotential gonad, meaning it possesses the capacity to differentiate into either female or male gonads. The next phase involves the development of a testis or ovary, which is entirely dependent upon the expression of the TDF gene SRY.

Initially, the gonads arise as paired structures within the intermediate mesoderm, where there are three parts that comprise the urogenital ridge: the pronephros (caudally), the mesonephros (central region where the gonad arises) and the metanephros (posteriorly, forming the kidney).
Cells that delaminate from the epithelium of the coelom provide a source of cells for the growing genital ridges and underlying cells from the mesonephros also expands the cell population in the gonadal primordia of males. Also, supporting cell precursors such as for Sertoli or Leydig cells are present within this early time period. The mesonephric ducts (Wolffian ducts) go on to form the ductal system of the male gonads and mesonephric tubules form shortly later, playing an important role in signaling surrounding areas for testis development.
Differentiation of testis occurs when the SRY gene is expressed within somatic cells, inducing them to form into Sertoli cells, which in turn, lead to the differentiation of all other cells present within the testis. Simultaneously, the gonad increases its size due to increased growth and movement of cells from the adjacent mesonephros. These cells give rise to peritubular myoid cells, endothelial cells that go on to form vasculature of the male gonad and to Leydig cells. The next stages involve testis-cord formation, Leydig cell formation (which secretes androgens required for fetal masculinization and the development of external genitalia). <ref><pubmed>17237341 </pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/17237341]</ref>

<references/>

'''Image: Diagram representing certain persistent portions of the mesonephros in the male'''
[[File:Bailey309.jpg|500px]]

==Online Assessment 8==
===Peer Reviews===
====Group Project 1====

''Respiratory Development''

The introduction is very informative and I particularly like how it describes the embryonic development of the respiratory system as well, since in order to understand what is happening in the fetal period, it is important to first understand what happened before that in the embryonic period. Perhaps the introduction could also introduce what information the page is going to contain.

The timeline is well presented in a table form, however maybe it would be better suited to be in the introduction section. The table could also incorporate the use of histological images to illustrate the differences between the time periods. Also, the sub sections titled ‘current models’ and ‘current research and findings’ could be part of a larger section and not fall under the ‘Lung Development Stages’ section.

There is no information as yet under ‘Current models’ however extensive research seems to be conducted on ‘current research findings’. Perhaps it would be better to include more journal articles in this section. The use of dot points and numbering systems is also very effective in allowing the information to be easily read and flow. More articles also need to be covered in the ‘Historic findings section’ as it is very brief at the moment with only a few sentences on each article.

The ‘abnormalities’ section is very well done with an abundance of conditions however more images should be uploaded for each abnormality in order to see what it visually presents as in the fetus and also to make the page look nicer.

The images uploaded onto the page contain adequate information explaining them, copyright information as well as the student image template, which is good. There is one student drawn image, which is also great, but maybe some more would further illustrate the group’s understanding of their topic.

The referencing is done correctly mostly throughout the page but is scattered throughout every section so perhaps it would be better to have them in one section at the bottom of the page under the heading entitled ‘References’ and numbered as they appear in the text. In-text citations are throughout and appear to be done correctly.

Overall, this is a very good effort and a bit of editing will make the page look much more neater and organized. Keep up the great work!

====Group Project 2====

''Renal Development''

The introduction provides a very informative description of the functions of the kidney and bladder. Perhaps it would be good to give some more details of the embryonic development just to quickly summarise what has been happening with the fetus up until this point. Also, maybe the introduction should introduce what the page’s content is going to cover. The order of historic findings and then developmental timeline is appropriate as historic findings can be used to compile the timeline. It would also be useful to have the timeline in a table format to make the page look neater and more simplified. Also, there is no research done on ‘historic findings’ so need to address that before final submission.

‘Current research models’ section is good but brief and requires more extensive research as only two articles are cited. There should be information on current models used to study renal development as well as current research and findings. The image in this section is well presented, with appropriate titling, referencing, image descriptions and copyright information with the student image template.
Sections 1.5-1.8 should be smaller sub headings under the larger heading ‘System Development’ and perhaps should go at the top of the page, beneath the introduction seeing as in order to understand research and historic findings, it is necessary to understand renal development first.

It is very good that there is a small section on early development, however maybe it would be better to have it more briefly explained, perhaps in the form of a student drawn diagram or presented as a table. There also is a problem with the image uploaded in the early development section, so should fix that before final submission. The ‘abnormalities’ section is also done well however more conditions should be listed and described with pictures for each one. There are also only abnormalities of the kidneys listed, so maybe it would be better to have more of the other components of the renal system as well (bladder, ureter, urethra).

Also, maybe more information regarding the anatomy of the kidneys and renal system should be added, as this is an anatomy course. Some images are also missing the student image template.
Most images are uploaded correctly with the right information, maybe more would make the page look more aesthetically pleasing as well as assist learning.

Referencing is done correctly with a numbering system and in-text citations are also correct. The in-text referencing in the ‘anatomical position’ sub section of ‘fetal development’ of the ‘Kidney’ section is not referenced appropriately so just fix that minor problem.

Overall, this is great work and should just include more information in certain sections and upload more images, preferably some student drawn images as well. Well done!

====Group Project 3====

''Gastrointestinal Development''

‘GIT system overview’ section is good but requires more information to introduce the GIT and what the page is going to have information on. Timeline could form part of this section and could also preferable be in the form of a student drawn image or even a table. The overview section also contains no in-text citations. It’s a great idea to split the GIT into the three parts: foregut, midgut and hindgut to aid in understanding. There is not much information on recent findings without any mention of current models as well so perhaps it would be best to address this before final submission.

In the foregut section there is not much mention of blood supply or innervation as was done for midgut and hindgut. Student drawn images are very impressive and referenced correctly with the student template, description, title and copyright information. The features of the midgut section could include some histological drawings or images. The ‘abnormalities’ section does not contain many in-text citations in one of the paragraphs and could include more deformities listed and described with more images, as well as information on how to treat and manage such disorders later in life. There is also no information or images addressing historical findings or current models so this needs to be looked into.

The references are correctly done and ordered, and are present at the bottom of the page. Some of the in-text citations aren’t throughout the text like they should be, for example, in the stomach, liver and gallbladder, and oesophagus sections.

Overall, good effort so far but more extensive research needs to be conducted for models and findings and more information for Abnormalities, as well as a few minor edits to make the page present more nicely.

====Group Project 5====

''Integumentary Development''

This page looks very neat and well organised, with an introduction that explains exactly what is going to appear and be discussed on the page. The Development Overview section is very well done, with the appropriate use of subheadings and content. The use of dot points is very effective, making the page look neater. Perhaps it would be good to draw a histological diagram of the skin layers, and uploading it to the skin development section. Specialised cells or important names throughout the page could be highlighted in bold or underlined as well, to highlight important terms and make it easier to learn and remember from. The title ‘Some Recent Findings’ accurately portrays what we as students can only do, which is identify SOME of the recent findings. This section could have more than 2 recent findings however and could be further subdivided by subheadings into the different components of the integumentary system – perhaps have 2-3 research articles for each component of the system. Historic findings are well researched but some more information would be good. The ‘Abnormalities’ section is so far the best looking section as it seems it is almost completed. Perhaps a few more abnormalities would be even better.

The table of the timeline in the ‘Development Overview’ section is superbly done and the use of histological images is fantastic as it provides the anatomical information visually. When I clicked on an image however, there was no proper referencing of the image and the copyright information and student image information was not present. The images are described very well.
One image has a problem and is present in red writing, so might need to remove this as something is wrong with the file and it could not be uploaded. There are no student-drawn images and I think if this group did this, it would really benefit their project and emphasise their understanding.

The ‘Some Recent Findings’ section has a purple background, which makes the page look more aesthetically pleasing and less monochrome. I like the ‘More recent papers’ box that can be expanded to reveal any more research papers related to the integumentary fetal development, in case anyone wants to have a further read- very clever.

Journal articles are correctly referenced but website references need to be improved upon- to find how to do this go to the ‘How to reference’ page. References are all over the place and need to be compiled under each heading or one main heading titled ‘References’ at the bottom of the page.

Overall, this page is looking fantastic at this point in time so keep up the great work!

====Group Project 6====

''Endocrine Development''

An introduction could be very useful to summarise what the page is going to discuss. Sections 1.2-1.11 could all be subheadings under the main heading ‘System Development’, and then each of these subheading could be further divided into smaller subheadings with timeline, introduction detailing structure/ function of the endocrine organ. It is however very well done how the headings of each organ are then further subdivided into ‘abnormalities’, ‘research findings’ and ‘timeline’. However, the fact that each section has its own references and is subdivided as such, shows that even though the page may appear more ordered, there appears to be little communication between group members at this stage. So perhaps a goal could be to make the page look like one flowing work piece as opposed to sections that each person has done.

I think the content is very well researched and I like the way each organ of the endocrine system is discussed, as all are important in fetal development. The use of images is appropriate and very well done as they are referenced correctly and when you click on an image it takes you to a new page showing the student image template, copyright information as well as extra information regarding the image. There are no student-drawn images however, so perhaps it could be possible to draw a flow chart perhaps of gonadal fetal development. The use of tables is also done very well and is frequent throughout the page, with some being used to illustrate the anatomical development of certain organs, for example, the adrenal gland and pancreas. The graphs are also useful in portraying information from research findings.

The project page is missing information regarding historic findings, and I think that if this page is going to have a main heading for Abnormalities, then the group should put all their information regarding abnormalities under this section. Although it is not an endocrine organ that grows within the developing foetus, but is an important part of the mother, there is not much information on the page regarding the placenta. This section needs to be completed as the placenta is an important source of hormones and acts as an endocrine organ during the pregnancy, sustaining the foetus.

It is good that there are many references, indicating thorough research into the endocrine system with each organ heading have its own sources, however I think these references need to be ordered better. The actual referencing is done correctly, however in-text referencing is absent, so it may be best to fix this. Most images are referenced correctly as well.

Overall, keep up the good work, but just edit the page to make it look neater and finish the sections you need to.

====Group Project 7====

''Neural (CNS) Development''

This project page is very nicely organised with the group clearly specifying what aspect of neural development they are covering, being the CNS. The use of headings and subheadings is done very neatly, however sections 1.1-1.5 could be subheadings for the larger title ‘system development’. The key points have been clearly described but there is no referencing throughout the ‘Introduction’, ‘Brain development’ and ‘Abnormalities’ sections. Most key points have at least some information on them which is good for this stage of the project; however some of the headings without could use some more work.

The choice of content is highly appropriate and the use of diagrams and pictures help show the groups understanding of the project thus far. I particularly like the use of subheadings in this project as they make the page look neater and organised. The image showing the timeline of fetal neural development is good however perhaps it would be better to draw or make a timeline on the computer in order to show better understanding of the time course of fetal development. Most images that have been uploaded are also well referenced and when clicking onto them, it takes the reader to a page that has more information related to the image. The table to describe anatomical details is also done well and is important that such a key point is mentioned seeing as this is an anatomy course.

I also really like how the ‘Current research, models and findings’ section is split into ‘Current research’ and ‘Future Research’, however it seems future research needs to be further looked into. The ‘Abnormalities’ section is done very well, with multiple abnormalities listed with images used to show each one. The bolding of several key words is seen and is helpful in showing understanding of some of the key points. There are also no historic findings so try and find some information on that.

Referencing is correctly done with most references being in one main section at the end, and ordered correctly. In-cite referencing is also done correctly. All images are correctly referenced with copyright information present and the student image template. I also like the way the current research findings sources have been referenced with the use of dot points assisting learning by not just presenting to the reader as a blob of information.

Overall, well done group 7! Keep up the great work!

====Group Project 8====

''Musculoskeletal Development''

The key points of musculoskeletal development appear as headings however there is still much that needs to be clearly discussed beneath each of these points. The main headings are good and specific but some are way too specific and should be under much larger headings, for example, 1.2-1.9 could be subheadings that come under the heading ‘System Development’. ‘Background embryonic development’ is useful to understand but perhaps it is better to not have so much detail, or summarise it in a table. The ‘Abnormalities’ heading is done well, with one disease listed (Duchenne Muscular Dystrophy). It might be better to have more than one abnormality listed and clearly described as well. I particularly like the use of statistics and genetic references. It seems most of the key points relating to system development have been clearly described, but some tidying up in terms of editing needs to be done.

Also, more work needs to be done on historic findings, current research, models and findings. Once all the research parts are completed, the timeline can be correctly constructed. Also like the idea of putting a timeline and the heading shows that this is intended. More subheadings could be used to make the page look more organised and pleasing to the eye.

There are also no graphs or tables as well as pictures. A table could be used to make the timeline or highlight the differences between the second, third trimesters and neonatal periods of fetal muscular development. Maybe the initial heading of the page should be changed to ‘Muscular Fetal Development’ to indicate that muscular development is actually being covered instead of both muscular and skeletal. There also isn’t much information regarding limb fetal development, so maybe it would be good to go through that on a deeper level.

It could also help to have images loaded onto the page or to draw flow diagrams to assist in the description of how the muscles develop in the fetal period. For example, upload an image showing the difference between slow twitch and fast twitch muscle fibres or draw a flow chart to show better understanding of the molecular and cellular regulation of fetal myogenesis.

References need to be in one larger section at the end under the heading ‘References’, not two and scattered throughout as is seen. The major section of references appears to be referenced correctly and in-cite references are done very well. There are also many references which are good and show that this group has thoroughly researched their topic.

Overall, this group has done very well and just needs to add more information for certain headings, as well as organise the page a bit better in neater headings and subheadings. Pictures should be added, as well as graphs, tables and own student-drawn images.

==Online Assessment 9==

'''Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos'''.<ref name=PMID22819672><pubmed>22819672</pubmed></ref>

One of the early stages of embryonic eye development involves the invagination of the optic vesicle resulting in the formation of the bilayered optic cup with a groove on its anterior aspect. This groove is termed the optic fissure (also known as embryonic fissure) and creates an opening into which the hyaloid artery and vein can enter and exit the developing eye. As time passes, this fissure begins to fuse back together, enclosing the hyaloid vessels and this event occurs between the 6th to 7th weeks of gestation<ref name=PMID1628748><pubmed>1628748</pubmed></ref>. The zebrafish is a suitable model for observing such development due to its faster time course, so in embryos the fissure takes approximately 2 days post-fertilisation to close. When this optic fissure fails to close, a disorder known as ocular coloboma occurs, leading to impaired vision and possibly blindness later on in life<ref name=PMID16932062><pubmed>16932062</pubmed></ref>.

Further studies conducted on zebrafish and mouse models have shown genetic mutations are responsible for the abnormal patterning of the optic vesicle and decreased gene expression involving the anterior eye and periocular mesenchyme and subsequently, excess tissue cell proliferation and fusion abnormalities<ref name=PMID17609112><pubmed>17609112</pubmed></ref>. Therefore, it is evident that there are many mechanisms responsible for the fusion event of the optic fissure but no studies have previously attempted to understand the mechanisms that result in the failure of this fusion event. Hence, the present study hypothesizes that the hyaloid vasculature is somehow related to this fusion event and if there are variations in the blood vessels such as dilatations, then the optic fissure does not close properly.

The study uses zebrafish Imo2 mutants that fail to close the optic fissure at 2 days post-fertilisation. This was done by isolating RNA from Imo2 mutants (1-day post-fertilisation) and cloning it using vectors. The gene mutation was then introduced into the RNA and injected into the embryo. Once the 2-day post-fertilisation window passed, embryos were fixed and tissues were sectioned and stained. In-situ hybridisation of the embryos was performed, antibodies labeled and TUNEL performed. On another set of embryos, microangiography and imaging was performed and data quantified and analysed.

The present study searched for mutations responsible for causing ocular coloboma using a genetic screen. A mutant line designated vu270 was identified at 2 days post-fertilisation, where failure of the fusion of the optic fissure was evident. Two other phenotypes of the embryos were observed, being a larger head and no apparent red blood cells. The zebrafish injected with the mutation as stated earlier failed to generate red blood cells and thus, this study proved that the lmo2 gene has a crucial role for hematopoiesis. The study also condemns the zebrafish an appropriate model to study the roles of lmo2 in embryonic development.
In comparison to its role in red blood cell formation, the functions of lmo2 in vascular development are not as well known. Whilst previous studies demonstrated the requirement of lmo2 in angiogenesis<ref name=PMID11857074><pubmed>11857074</pubmed></ref>, the present study although reiterated this, showed abnormal formation of the blood vessels. This study further condemned the zebrafish as a relevant model to study the function of lmo2 in angiogenesis and the abnormalities associated with the vasculature that arises due to this gene, are a result of the genes’ role in maintaining vascular permeability and integrity. Therefore, it is evident that the absence of the lmo2 gene correlates with increased permeability of the vasculature.
Even more specifically, the results showed constrictions within the hyaloid artery and nerve, but severe dilatations in the hyaloid vein, indicating that lmo2 has different roles in the development of different blood vessels, or that in constricted vessels there is no flow. However, the idea that in constricted blood vessels there is no flow can be rid of as a conclusion since microangiography results showed blood flow.

In conclusion, the results of the present study demonstrate that abnormal blood vessels coursing through the optic fissure can indeed interfere with its closure.
[[Sensory - Vision Development]]

<references/>

==Online Assessment 10==

<pubmed>25085858</pubmed>

Due to the increased incidence and mortality rates of cardiovascular diseases such as ischaemic heart disease, aortic aneurysms and peripheral vascular diseases in Western society, such conditions have called for various treatment options, including vascular bypass grafting and replacements.<ref name=PMID16397155><pubmed>16397155</pubmed></ref> Much of the time, patients do not have suitable corresponding vessels for such bypass treatments or replacements and sometimes even result in thrombosis, infection and pseudoaneurysms.<ref name=PMID23197861><pubmed>23197861</pubmed></ref>This is of particular importance in the paediatric field, where children require multiple surgeries to accommodate for the lack of growth of the vasculature and synthetic vascular grafts. <ref name=PMID19099046><pubmed>19099046</pubmed></ref> Therefore, there is a great need for new and improved vascular grafts and vascular replacement treatment options and such a need has been the driving force for the development of tissue-engineered blood vessels. Pluripotent stem cells have developed into a promising source of cells due to their high proliferating capacity and high differentiation potential to form different types of cells. Previous research successfully differentiated embryonic stem cells and induced pluripotent stem cells from mice into smooth muscle cells, indicating their potential for use in regenerative medicine and vascular engineering.<ref name=PMID21439638><pubmed>21439638</pubmed></ref>
This study aims to create human induced pluripotent stem cells from patient primary aortic fibroblasts and turn them into functional smooth muscle cells. It also examines the ability of the derived smooth muscle cells to construct vascular tissues on predesigned three-dimensional biodegradable scaffolds.

The study isolates and cultures primary aortic fibroblasts from a heart transplant donor, by sterilizing the tissue, removing the tunica intima and separating the tunic media and adventitia. The tunica media and adventitia were then treated with various chemicals to wash out the fibroblasts and smooth muscle cells. Induced pluripotent stem cell lines were then generated and characterized, and then stem cells were then allowed to differentiate into smooth muscle cells. The muscle cells were then assayed based on their contractility and quantitative RT-PCR, flow cytometry, fabrications of 3D scaffolds, and construction of tissue-engineered vascular tissues was performed. Scanning electron microscopy, histological observations and statistical analyses were also conducted.

The study successfully isolated the fibroblasts from the primary aortic tissue and induced pluripotent stem cell lines could be maintained and expanded on MEF feeders and Matrigel-coated surfaces. Many of these stem cell lines maintained their pluripotency, evident by RT-PCR results showing SOX2, NANOG and OCT4 transcription factors. The study also found that using a smaller pore size within the macroporous scaffold was better able to support the smooth muscle cell proliferation, resulting in a higher smooth muscle cell density.

In conclusion, construction of a whole tissue-engineered blood vessel requires the addition of all the other different layers, and not just smooth muscles cells, for example, cells from the tunica intima, media and adventitia. Future studies will involve the culturing of these other cell types altogether and could have even greater implications for future regenerative medicine.

<references/>

User talk:Z3417753

2014-10-27T09:47:05Z

Z3417753:

[http://www.ncbi.nlm.nih.gov/pubmed PubMed]

[http://www.ncbi.nlm.nih.gov/pubmed/25084016 PMID25084016]

<pubmed>25084016</pubmed>

[http://www.plosone.org/ PLOS ONE]

<pubmed>23227157</pubmed>

* Currently there are no effective treatments for children suffering with advanced anatomical disorders of the trachea.

* An example of such a disorder is congenital tracheal stenosis, characterised by the presence of complete tracheal rings and stenosis of the larger airways.<ref name=PMID22381446><pubmed>22381446</pubmed></ref>

* Children suffering with such congenital abnormalities often require constant hospitalisation with slide tracheoplasty being the preferred choice of treatment, however it is very common for patients to develop recurrent stenoses.<ref name=PMID15573072><pubmed>15573072</pubmed></ref>

* This is often a result of stent erosion<ref name=PMID11053808><pubmed>11053808</pubmed></ref>, ultimately leading to death or the ability of the trachea to undergo somatic growth.<ref name=PMID22381446><pubmed>22381446</pubmed></ref>

* Due to the severity of these conditions, they are considered fatal and once detected prenatally, the pregnancies are often terminated.<ref name=PMID21860060><pubmed>21860060</pubmed></ref>

* In the past few years, there has been an increased use of tissue-engineered structures made from stem cells within clinical settings.

* Benefits of stem cell-based tissue engineering procedures arise from the notion that remodeling of the native stroma would result in a lack of immunosuppression due to the similarity of the local tissue.

* A case was reported where a successful stem cell-based tracheal replacement procedure was performed on a 26-year-old patient with end-stage airway disease.<ref name=PMID19022496><pubmed>19022496</pubmed></ref>

* Whilst stem cell-based tracheal grafts have been successful with positive short-term outcomes, little evidence exists regarding the long-term effects of the grafts, specifically on children.

* This study reports a 2-year follow-up of the first paediatric stem cell-based tracheal replacement, with the notion in mind that ideal results following the procedure are:

*: Normal airway and lung function
*: Appropriate somatic growth
*: Increased quality of life
*: Elimination of the need for continuous hospitalisation and the constant advent of surgical procedures.

<references/>

User talk:Z3417753

2014-10-27T09:45:39Z

Z3417753:

[http://www.ncbi.nlm.nih.gov/pubmed PubMed]

[http://www.ncbi.nlm.nih.gov/pubmed/25084016 PMID25084016]

<pubmed>25084016</pubmed>

[http://www.plosone.org/ PLOS ONE]

<pubmed>23227157</pubmed>

* Currently there are no effective treatments for children suffering with advanced anatomical disorders of the trachea.

* An example of such a disorder is congenital tracheal stenosis, characterised by the presence of complete tracheal rings and stenosis of the larger airways.<ref name=PMID22381446><pubmed>22381446</pubmed></ref>

* Children suffering with such congenital abnormalities often require constant hospitalisation with slide tracheoplasty being the preferred choice of treatment, however it is very common for patients to develop recurrent stenoses.<ref name=PMID15573072><pubmed>15573072</pubmed></ref>

* This is often a result of stent erosion<ref name=PMID11053808><pubmed>11053808</pubmed></ref>, ultimately leading to death or the ability of the trachea to undergo somatic growth.<ref name=PMID22381446><pubmed>22381446</pubmed></ref>

* Due to the severity of these conditions, they are considered fatal and once detected prenatally, the pregnancies are often terminated.<ref name=PMID21860060><pubmed>21860060</pubmed></ref>

* In the past few years, there has been an increased use of tissue-engineered structures made from stem cells within clinical settings.

* Benefits of stem cell-based tissue engineering procedures arise from the notion that remodeling of the native stroma would result in a lack of immunosuppression due to the similarity of the local tissue.

* A case was reported where a successful stem cell-based tracheal replacement procedure was performed on a 26-year-old patient with end-stage airway disease.<ref name=PMID19022496><pubmed>19022496</pubmed></ref>

* Whilst stem cell-based tracheal grafts have been successful with positive short-term outcomes, little evidence exists regarding the long-term effects of the grafts, specifically on children.

* This study reports a 2-year follow-up of the first paediatric stem cell-based tracheal replacement, with the notion in mind that ideal results following the procedure are:

** Normal airway and lung function
** Appropriate somatic growth
** Increased quality of life
** Elimination of the need for continuous hospitalisation and the constant advent of surgical procedures.

User:Z3417753

2014-10-26T05:33:58Z

Z3417753: /* Online Assessment 10 */

{{StudentPage2014}}

==Lab Attendance==
* Lab 1 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 12:54, 6 August 2014 (EST)
* Lab 2 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:21, 13 August 2014 (EST)
* Lab 3 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 20 August 2014 (EST)
* Lab 4 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:45, 27 August 2014 (EST)
* Lab 5 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:48, 3 September 2014 (EST)
* Lab 6 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:50, 10 September 2014 (EST)
* Lab 7 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:31, 17 September 2014 (EST)
* Lab 8 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 24 September 2014 (EST)
* Lab 9 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:27, 8 October 2014 (EST)
* Lab 10 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:11, 15 October 2014 (EST)
* Lab 11 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:26, 22 October 2014 (EST)

==Online Assessment 1==

===Article 1===
<pubmed>23148203</pubmed>

This study is an analysis of the optimal time from oocyte to preimplantation embryo development for biopsy and preimplantation genetic screening. The discovery of the optimal time can then be used to detect any abnormal chromosomal separation patterns in embryos from older mothers (>40 years old). The study was a longitudinal cohort study involving 9 infertile couples and 21 sets of complete chromosomal screening data, including polar bodies 1 + 2 and their corresponding blastomeres and trophectoderm samples.

METHODS →infertile couples with a good response to controlled ovarian stimulation were enrolled in the study and underwent IVF. Polar bodies, blastomeres and trophectoderm samples were biopsied and analysed by array comparative genomic hybridisation. The chromosomal segregation patterns were analysed from these results and used to deduce the origin of aneuploidy. The results were also used to examine the accuracy of polar body and cleavage-stage preimplantation genetic screening strategies.

RESULTS → Since preimplantation genetic screening tests have been conducted at different times throughout the preimplantation window, it is possible that critical bits of information regarding chromosomal segregation patterns have been missed. Thus, by performing such tests at an optimal time, we are better able to understand these meiotic chromosomal segregation patterns and therefore potentially increase the success rates of in-vitro fertilisation. This study uses a sequential chromosome analysis of polar bodies and their corresponding embryos at both the cleavage and blastocyst stages in order to work out what stage is best to perform these genetic screening tests and biopsies, potentially increasing IVF success rate. The study showed that testing at the polar body stage was least accurate due to the high incidence of post-zygotic events and discovered that performing these tests later on in development (at the blastocyst stage) may produce more reliable results for the screenings, thereby achieving better chromosomal segregation pattern data. These results can now go on to be used for IVF research.

===Article 2===

<pubmed>23477909</pubmed>

This study examines the accuracy of using array comparative genomic hybridisation (array CGH) techniques for the analysis of first and second polar bodies in predicting aneuploidies of maternal meiotic origin in the cleavage stage embryos of women of advanced maternal age. It is known that aneuploidy is a common cause of pregnancy failure, miscarriage and abnormal pregnancy and most aneuploidy is due to maternal meiotic origin and increases exponentially as the mother approaches menopause.

METHOD → 20 couples requesting preimplantation genetic screening for advanced maternal age (=greater than or equal to 35 years old) and repeated implantation failure (more than 3 cycles), previous aneuploidy pregnancy or recurrent first trimester miscarriage underwent 16 controlled ovarian hyperstimulation cycles and 7 natural fresh cycles. Male partners had sperm parameters within the normal range except for 2 which had oligoasthenoteratozoospermia. Oocytes were retrieved by ultrasound-guided transvaginal aspiration 36 hours after beta-hCG administration. Once the oocytes were retrieved, biopsy of the first polar body was performed and the oocyte was inseminated using intracytoplasmic sperm injection. The following morning, each oocyte was checked for prouclei and extrusion of the second polar body to confirm fertilisation. The second polar body was then biopsied. The polar bodies were then analysed using array CGH analysis and the zona pellucida layer of the oocyte was dissolved. The zona-free embryo then underwent whole genome amplification and array CGH analysis in the cleavage stage.

RESULTS → It has been demonstrated in previous studies that a high correlation exists between the chromosomal status presented from polar body analysis and the actual chromosomes present in the zygotes of older mothers. Due to these results, this study uses polar body analysis and array CGH analysis of mature fertilised oocytes, to identify errors in meiosis within the polar bodies as well as the corresponding cleavage stage embryos. The results of the current study showed that nearly ALL aneuploidies detected in cleavage stage embryos were associated with copy number changes in the polar bodies (93%), indicating the high capability of polar bodies being used to predict aneuploidy and what is actually happening within the embryo.

--[[User:Z8600021|Mark Hill]] These are good articles and summaries. Reference link is formatted correctly (5/5)

==Online Assessment 2==
[[File:Fusion of two pairs of blastomeres inside 4-cell embryos.png|800px]]

Fusion of two pairs of blastomeres inside 4-cell embryos<ref><pubmed>23227157</pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515590/figure/pone-0050029-g003/]</ref>

<references/>

--[[User:Z8600021|Mark Hill]] Image appropriate for assessment and all associated information formatted correctly. You may want to include species (mouse) information with the image. (5/5)

==Online Assessment 3==
===Current Research Models and Findings===
--[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 22:57, 26 August 2014 (EST)

<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>

--[[User:Z8600021|Mark Hill]] These are relevant references, a slingle line describing why you have selected these would have been also useful to include. (4/5)

==Online Assessment 4==
===Cord Stem Cell Article Findings===

<pubmed>25101638</pubmed>

This article identifies acute liver failure as a devastating and debilitating illness that occurs within a short period of time, ultimately resulting in death of the patient if proper treatment is unavailable or it is simply too late to treat. It further identifies liver transplantation as the most effective treatment to date however, its application is limited due to an elevated risk of organ rejection and lack of liver donors. It is also known that human umbilical mesenchymal stem cells (hUCMSC) have the potential to differentiate into hepatocyte-like cells, functioning very similarly to hepatocytes as well as secrete certain factors to stimulate the proliferation of nearby hepatocytes, thereby promoting the rejuvenation of the host liver cells. The author hypothesised that by decreasing the amount of manipulation received by the mesenchymal stem cells in vitro, the carcinogenic risk was reduced. As a result, the therapeutic effect (amount of liver repair) of concurrently acting hUCMSC’s and hepatocyte-like cells can be ascertained by studying and comparing the two synchronous actions in acute liver failure mouse models.

The study induced acute liver failure in mouse models using D-galactosamine and lipopolysaccharide, causing the death of approximately 50% of the mice (necrosis of more than 50% of the hepatocytes). The mouse models’ therapeutic effects were then compared before and after the mesenchymal stem cells were differentiated into hepatocyte-like cells, by transplanting and injecting the cells into the tail vein. The results showed that almost ALL mouse were saved by the injection of the hepatocyte-like cells. Similarly, the injection of the hUCMSC’s also demonstrated their capability to repair liver damage, however, the population of these cells tested via the expression/ presence of human hepatocyte growth factor was minimal, suggesting that they allow the reversal of acute liver failure by differentiating into hepatocyte-like cells.

Overall, these results suggest that hUCMSC’s and hepatocyte-like cells are just as effective in therapeutic treatment of acute liver failure in mouse models and that hUCMSC’s play a larger role in stimulating the host hepatocyte repair.

===Vascular Shunts===

Three major vascular shunts exist within the circulatory system of the foetus:

1. FORAMEN OVALE --> the opening in the interatrial septum (wall between left and right atrium) that allows the flow of blood from the right atrium to the left atrium and has a valve to prevent backflow during the fetal period. It soon closes once right atrial pressure increases.
The foramen ovalis then becomes the FOSSA OVALIS postnatally.

2. DUCTUS ARTERIOSUS --> muscular vessel that connects the pulmonary trunk to the aorta, thereby diverting bloodflow to the lungs and going straight into the aorta. After birth, as the amount of oxygen increases, the smooth muscle in the walls constricts closing off the passage.
As the ductus arteriosus degenerates, all that is left behind if the LIGAMENTUM ARTERIOSUM.

3. DUCTUS VENOSUS --> a blood vessel that branches from the umbilical vein, allowing oxygenated blood from the placenta to be diverted from the fetal liver to the fetal heart.
This shunt closes slowly during infancy and degenerates into the LIGAMENTUM VENOSUM.

==Online Assessment 5==
Laryngeal-tracheo-oesophageal Cleft is a rare congenital anomaly where there is an abnormal posterior communication between the larynx and pharynx, extending down between the trachea and oesophagus.

Normally, the larynx develops simultaneously from the endoderm (arising from the foregut region) and the mesenchyme (arising from the 4th + 6th pharyngeal arches. The division of the foregut is due to the fusion of the lateral walls of the foregut in the region of the larynx, thereby forming a septum that divides the foregut into a central part = LARYNGEAL-TRACHEAL TUBE as well as a dorsal portion = OESOPHAGUS. The mesenchymal portion (= TRACHEAL-OESOPHAGEAL SEPTUM) is located between the digestive and respiratory tracts and is the result of the separation of the two tracts. Apoptotic epithelial cells are also present at this septum, mainly in the ventral portion, but inactive in the dorsal portion.

There are a few models that explain tracheal-oesophageal anomalies, including Laryngeal-tracheo-oesophageal Cleft:

• INTRAEMBRYONIC PRESSURE → an intense curvature of the cervical region during heart development places pressure upon the oesophagus and as a result displaces it, leading to growth abnormalities.

• EPITHELIAL OCCLUSION → the oesophagus is solid during a stage of development but it is soon recanalised. If the recanalisation does not occur, growth abnormalities may occur.

• VASCULAR OCCLUSION → an abnormally communicating vessel could lead to avascularisation in the foregut, resulting in abnormalities. In the case of Laryngeal-tracheo-oesophageal Cleft, this means the laryngeal region.

• DIFFERENTIAL CELL GROWTH → abnormal cell growth in the ventral or dorsal part of the developing trachea or oesophagus could result in defects of the two tracts.<ref><pubmed>22151899</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22151899]</ref>

<references/>

==Online Assessment 6==

===Research Article on Development of the Pancreas===
<pubmed>24375815</pubmed>

This article explores the role of pancreas-specific transcription factor 1a (PTF1a) in development of the pancreas, but makes a note of the difficulty associated with obtaining embryonic tissue specimens for experimentation. Due to the limitations associated with such embryonic material, embryonic stem cells (ESCs), which can be differentiated in vitro are used as a model system to study and examine the role of PTF1a in the development of the pancreas.
The study uses cell cultures, quantitative PCR, immunofluorescent staining, flow cytometry and western blot staining to demonstrate that PTF1a is required very early in development for arrangement of the pancreas from the foregut endoderm. The study shows that PTF1a drives differentiation of pancreatic cells from embryonic stem cells and this determines PTF1a to be an initiator of pancreatic differentiation in the form of ductal, endocrine and exocrine cells. The ectopic expression of PTF1a stimulated the ESCs to start differentiating into pancreas, causing the cells to activate PDX1 expression in bud-like structures that looked like early pancreas in vivo. The study also found that retinoic acid and nicotinamide signaling could regulate the ratio of endocrine to exocrine cell differentiation.
The future implications of this study may involve further studies utilizing the notion of the importance of activating PTF1a in the development of enhanced pancreatic differentiation for creating ESC-derived insulin (beta cells) expressing cells.

===Embryonic Layers and Tissues that Contribute to Tooth Development===

Teeth are part of the integumentary system and are formed by epithelial as well as mesenchymal interactions during development. They are largely formed by ECTODERM of the first pharyngeal arch, MESODERM and receive a major contribution from NEURAL CREST ECTOMESENCHYMAL cells. The neural crest mesenchymal cells change due to the enamel epithelium.

==Online Assessment 7==

'''TIME COURSE OF EMBRYONIC DEVELOPMENT OF THE HUMAN TESTIS'''

During fertilisation, genes determine the sexual fate of the organism and whether the organism is male or female is only revealed in fetal development when development of the external genitalia finally occurs. The presence of the Y chromosome leads to the development of testes in humans and their development is dependent on a single gene located on this Y chromosome known as Testis-determining factor (TDF).

When observing the morphology and cell biology of the developing testis, it is important to note that much of the research conducted on the subject involved the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy.

The next stage in embryonic development of the human testis is the formation of the genital ridge in an initial phase. This genital ridge is representative of the ambisexual stage in human embryos and is a bipotential gonad, meaning it possesses the capacity to differentiate into either female or male gonads. The next phase involves the development of a testis or ovary, which is entirely dependent upon the expression of the TDF gene SRY.

Initially, the gonads arise as paired structures within the intermediate mesoderm, where there are three parts that comprise the urogenital ridge: the pronephros (caudally), the mesonephros (central region where the gonad arises) and the metanephros (posteriorly, forming the kidney).
Cells that delaminate from the epithelium of the coelom provide a source of cells for the growing genital ridges and underlying cells from the mesonephros also expands the cell population in the gonadal primordia of males. Also, supporting cell precursors such as for Sertoli or Leydig cells are present within this early time period. The mesonephric ducts (Wolffian ducts) go on to form the ductal system of the male gonads and mesonephric tubules form shortly later, playing an important role in signaling surrounding areas for testis development.
Differentiation of testis occurs when the SRY gene is expressed within somatic cells, inducing them to form into Sertoli cells, which in turn, lead to the differentiation of all other cells present within the testis. Simultaneously, the gonad increases its size due to increased growth and movement of cells from the adjacent mesonephros. These cells give rise to peritubular myoid cells, endothelial cells that go on to form vasculature of the male gonad and to Leydig cells. The next stages involve testis-cord formation, Leydig cell formation (which secretes androgens required for fetal masculinization and the development of external genitalia). <ref><pubmed>17237341 </pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/17237341]</ref>

<references/>

'''Image: Diagram representing certain persistent portions of the mesonephros in the male'''
[[File:Bailey309.jpg|500px]]

==Online Assessment 8==
===Peer Reviews===
====Group Project 1====

''Respiratory Development''

The introduction is very informative and I particularly like how it describes the embryonic development of the respiratory system as well, since in order to understand what is happening in the fetal period, it is important to first understand what happened before that in the embryonic period. Perhaps the introduction could also introduce what information the page is going to contain.

The timeline is well presented in a table form, however maybe it would be better suited to be in the introduction section. The table could also incorporate the use of histological images to illustrate the differences between the time periods. Also, the sub sections titled ‘current models’ and ‘current research and findings’ could be part of a larger section and not fall under the ‘Lung Development Stages’ section.

There is no information as yet under ‘Current models’ however extensive research seems to be conducted on ‘current research findings’. Perhaps it would be better to include more journal articles in this section. The use of dot points and numbering systems is also very effective in allowing the information to be easily read and flow. More articles also need to be covered in the ‘Historic findings section’ as it is very brief at the moment with only a few sentences on each article.

The ‘abnormalities’ section is very well done with an abundance of conditions however more images should be uploaded for each abnormality in order to see what it visually presents as in the fetus and also to make the page look nicer.

The images uploaded onto the page contain adequate information explaining them, copyright information as well as the student image template, which is good. There is one student drawn image, which is also great, but maybe some more would further illustrate the group’s understanding of their topic.

The referencing is done correctly mostly throughout the page but is scattered throughout every section so perhaps it would be better to have them in one section at the bottom of the page under the heading entitled ‘References’ and numbered as they appear in the text. In-text citations are throughout and appear to be done correctly.

Overall, this is a very good effort and a bit of editing will make the page look much more neater and organized. Keep up the great work!

====Group Project 2====

''Renal Development''

The introduction provides a very informative description of the functions of the kidney and bladder. Perhaps it would be good to give some more details of the embryonic development just to quickly summarise what has been happening with the fetus up until this point. Also, maybe the introduction should introduce what the page’s content is going to cover. The order of historic findings and then developmental timeline is appropriate as historic findings can be used to compile the timeline. It would also be useful to have the timeline in a table format to make the page look neater and more simplified. Also, there is no research done on ‘historic findings’ so need to address that before final submission.

‘Current research models’ section is good but brief and requires more extensive research as only two articles are cited. There should be information on current models used to study renal development as well as current research and findings. The image in this section is well presented, with appropriate titling, referencing, image descriptions and copyright information with the student image template.
Sections 1.5-1.8 should be smaller sub headings under the larger heading ‘System Development’ and perhaps should go at the top of the page, beneath the introduction seeing as in order to understand research and historic findings, it is necessary to understand renal development first.

It is very good that there is a small section on early development, however maybe it would be better to have it more briefly explained, perhaps in the form of a student drawn diagram or presented as a table. There also is a problem with the image uploaded in the early development section, so should fix that before final submission. The ‘abnormalities’ section is also done well however more conditions should be listed and described with pictures for each one. There are also only abnormalities of the kidneys listed, so maybe it would be better to have more of the other components of the renal system as well (bladder, ureter, urethra).

Also, maybe more information regarding the anatomy of the kidneys and renal system should be added, as this is an anatomy course. Some images are also missing the student image template.
Most images are uploaded correctly with the right information, maybe more would make the page look more aesthetically pleasing as well as assist learning.

Referencing is done correctly with a numbering system and in-text citations are also correct. The in-text referencing in the ‘anatomical position’ sub section of ‘fetal development’ of the ‘Kidney’ section is not referenced appropriately so just fix that minor problem.

Overall, this is great work and should just include more information in certain sections and upload more images, preferably some student drawn images as well. Well done!

====Group Project 3====

''Gastrointestinal Development''

‘GIT system overview’ section is good but requires more information to introduce the GIT and what the page is going to have information on. Timeline could form part of this section and could also preferable be in the form of a student drawn image or even a table. The overview section also contains no in-text citations. It’s a great idea to split the GIT into the three parts: foregut, midgut and hindgut to aid in understanding. There is not much information on recent findings without any mention of current models as well so perhaps it would be best to address this before final submission.

In the foregut section there is not much mention of blood supply or innervation as was done for midgut and hindgut. Student drawn images are very impressive and referenced correctly with the student template, description, title and copyright information. The features of the midgut section could include some histological drawings or images. The ‘abnormalities’ section does not contain many in-text citations in one of the paragraphs and could include more deformities listed and described with more images, as well as information on how to treat and manage such disorders later in life. There is also no information or images addressing historical findings or current models so this needs to be looked into.

The references are correctly done and ordered, and are present at the bottom of the page. Some of the in-text citations aren’t throughout the text like they should be, for example, in the stomach, liver and gallbladder, and oesophagus sections.

Overall, good effort so far but more extensive research needs to be conducted for models and findings and more information for Abnormalities, as well as a few minor edits to make the page present more nicely.

====Group Project 5====

''Integumentary Development''

This page looks very neat and well organised, with an introduction that explains exactly what is going to appear and be discussed on the page. The Development Overview section is very well done, with the appropriate use of subheadings and content. The use of dot points is very effective, making the page look neater. Perhaps it would be good to draw a histological diagram of the skin layers, and uploading it to the skin development section. Specialised cells or important names throughout the page could be highlighted in bold or underlined as well, to highlight important terms and make it easier to learn and remember from. The title ‘Some Recent Findings’ accurately portrays what we as students can only do, which is identify SOME of the recent findings. This section could have more than 2 recent findings however and could be further subdivided by subheadings into the different components of the integumentary system – perhaps have 2-3 research articles for each component of the system. Historic findings are well researched but some more information would be good. The ‘Abnormalities’ section is so far the best looking section as it seems it is almost completed. Perhaps a few more abnormalities would be even better.

The table of the timeline in the ‘Development Overview’ section is superbly done and the use of histological images is fantastic as it provides the anatomical information visually. When I clicked on an image however, there was no proper referencing of the image and the copyright information and student image information was not present. The images are described very well.
One image has a problem and is present in red writing, so might need to remove this as something is wrong with the file and it could not be uploaded. There are no student-drawn images and I think if this group did this, it would really benefit their project and emphasise their understanding.

The ‘Some Recent Findings’ section has a purple background, which makes the page look more aesthetically pleasing and less monochrome. I like the ‘More recent papers’ box that can be expanded to reveal any more research papers related to the integumentary fetal development, in case anyone wants to have a further read- very clever.

Journal articles are correctly referenced but website references need to be improved upon- to find how to do this go to the ‘How to reference’ page. References are all over the place and need to be compiled under each heading or one main heading titled ‘References’ at the bottom of the page.

Overall, this page is looking fantastic at this point in time so keep up the great work!

====Group Project 6====

''Endocrine Development''

An introduction could be very useful to summarise what the page is going to discuss. Sections 1.2-1.11 could all be subheadings under the main heading ‘System Development’, and then each of these subheading could be further divided into smaller subheadings with timeline, introduction detailing structure/ function of the endocrine organ. It is however very well done how the headings of each organ are then further subdivided into ‘abnormalities’, ‘research findings’ and ‘timeline’. However, the fact that each section has its own references and is subdivided as such, shows that even though the page may appear more ordered, there appears to be little communication between group members at this stage. So perhaps a goal could be to make the page look like one flowing work piece as opposed to sections that each person has done.

I think the content is very well researched and I like the way each organ of the endocrine system is discussed, as all are important in fetal development. The use of images is appropriate and very well done as they are referenced correctly and when you click on an image it takes you to a new page showing the student image template, copyright information as well as extra information regarding the image. There are no student-drawn images however, so perhaps it could be possible to draw a flow chart perhaps of gonadal fetal development. The use of tables is also done very well and is frequent throughout the page, with some being used to illustrate the anatomical development of certain organs, for example, the adrenal gland and pancreas. The graphs are also useful in portraying information from research findings.

The project page is missing information regarding historic findings, and I think that if this page is going to have a main heading for Abnormalities, then the group should put all their information regarding abnormalities under this section. Although it is not an endocrine organ that grows within the developing foetus, but is an important part of the mother, there is not much information on the page regarding the placenta. This section needs to be completed as the placenta is an important source of hormones and acts as an endocrine organ during the pregnancy, sustaining the foetus.

It is good that there are many references, indicating thorough research into the endocrine system with each organ heading have its own sources, however I think these references need to be ordered better. The actual referencing is done correctly, however in-text referencing is absent, so it may be best to fix this. Most images are referenced correctly as well.

Overall, keep up the good work, but just edit the page to make it look neater and finish the sections you need to.

====Group Project 7====

''Neural (CNS) Development''

This project page is very nicely organised with the group clearly specifying what aspect of neural development they are covering, being the CNS. The use of headings and subheadings is done very neatly, however sections 1.1-1.5 could be subheadings for the larger title ‘system development’. The key points have been clearly described but there is no referencing throughout the ‘Introduction’, ‘Brain development’ and ‘Abnormalities’ sections. Most key points have at least some information on them which is good for this stage of the project; however some of the headings without could use some more work.

The choice of content is highly appropriate and the use of diagrams and pictures help show the groups understanding of the project thus far. I particularly like the use of subheadings in this project as they make the page look neater and organised. The image showing the timeline of fetal neural development is good however perhaps it would be better to draw or make a timeline on the computer in order to show better understanding of the time course of fetal development. Most images that have been uploaded are also well referenced and when clicking onto them, it takes the reader to a page that has more information related to the image. The table to describe anatomical details is also done well and is important that such a key point is mentioned seeing as this is an anatomy course.

I also really like how the ‘Current research, models and findings’ section is split into ‘Current research’ and ‘Future Research’, however it seems future research needs to be further looked into. The ‘Abnormalities’ section is done very well, with multiple abnormalities listed with images used to show each one. The bolding of several key words is seen and is helpful in showing understanding of some of the key points. There are also no historic findings so try and find some information on that.

Referencing is correctly done with most references being in one main section at the end, and ordered correctly. In-cite referencing is also done correctly. All images are correctly referenced with copyright information present and the student image template. I also like the way the current research findings sources have been referenced with the use of dot points assisting learning by not just presenting to the reader as a blob of information.

Overall, well done group 7! Keep up the great work!

====Group Project 8====

''Musculoskeletal Development''

The key points of musculoskeletal development appear as headings however there is still much that needs to be clearly discussed beneath each of these points. The main headings are good and specific but some are way too specific and should be under much larger headings, for example, 1.2-1.9 could be subheadings that come under the heading ‘System Development’. ‘Background embryonic development’ is useful to understand but perhaps it is better to not have so much detail, or summarise it in a table. The ‘Abnormalities’ heading is done well, with one disease listed (Duchenne Muscular Dystrophy). It might be better to have more than one abnormality listed and clearly described as well. I particularly like the use of statistics and genetic references. It seems most of the key points relating to system development have been clearly described, but some tidying up in terms of editing needs to be done.

Also, more work needs to be done on historic findings, current research, models and findings. Once all the research parts are completed, the timeline can be correctly constructed. Also like the idea of putting a timeline and the heading shows that this is intended. More subheadings could be used to make the page look more organised and pleasing to the eye.

There are also no graphs or tables as well as pictures. A table could be used to make the timeline or highlight the differences between the second, third trimesters and neonatal periods of fetal muscular development. Maybe the initial heading of the page should be changed to ‘Muscular Fetal Development’ to indicate that muscular development is actually being covered instead of both muscular and skeletal. There also isn’t much information regarding limb fetal development, so maybe it would be good to go through that on a deeper level.

It could also help to have images loaded onto the page or to draw flow diagrams to assist in the description of how the muscles develop in the fetal period. For example, upload an image showing the difference between slow twitch and fast twitch muscle fibres or draw a flow chart to show better understanding of the molecular and cellular regulation of fetal myogenesis.

References need to be in one larger section at the end under the heading ‘References’, not two and scattered throughout as is seen. The major section of references appears to be referenced correctly and in-cite references are done very well. There are also many references which are good and show that this group has thoroughly researched their topic.

Overall, this group has done very well and just needs to add more information for certain headings, as well as organise the page a bit better in neater headings and subheadings. Pictures should be added, as well as graphs, tables and own student-drawn images.

==Online Assessment 9==

'''Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos'''.<ref name=PMID22819672><pubmed>22819672</pubmed></ref>

One of the early stages of embryonic eye development involves the invagination of the optic vesicle resulting in the formation of the bilayered optic cup with a groove on its anterior aspect. This groove is termed the optic fissure (also known as embryonic fissure) and creates an opening into which the hyaloid artery and vein can enter and exit the developing eye. As time passes, this fissure begins to fuse back together, enclosing the hyaloid vessels and this event occurs between the 6th to 7th weeks of gestation<ref name=PMID1628748><pubmed>1628748</pubmed></ref>. The zebrafish is a suitable model for observing such development due to its faster time course, so in embryos the fissure takes approximately 2 days post-fertilisation to close. When this optic fissure fails to close, a disorder known as ocular coloboma occurs, leading to impaired vision and possibly blindness later on in life<ref name=PMID16932062><pubmed>16932062</pubmed></ref>.

Further studies conducted on zebrafish and mouse models have shown genetic mutations are responsible for the abnormal patterning of the optic vesicle and decreased gene expression involving the anterior eye and periocular mesenchyme and subsequently, excess tissue cell proliferation and fusion abnormalities<ref name=PMID17609112><pubmed>17609112</pubmed></ref>. Therefore, it is evident that there are many mechanisms responsible for the fusion event of the optic fissure but no studies have previously attempted to understand the mechanisms that result in the failure of this fusion event. Hence, the present study hypothesizes that the hyaloid vasculature is somehow related to this fusion event and if there are variations in the blood vessels such as dilatations, then the optic fissure does not close properly.

The study uses zebrafish Imo2 mutants that fail to close the optic fissure at 2 days post-fertilisation. This was done by isolating RNA from Imo2 mutants (1-day post-fertilisation) and cloning it using vectors. The gene mutation was then introduced into the RNA and injected into the embryo. Once the 2-day post-fertilisation window passed, embryos were fixed and tissues were sectioned and stained. In-situ hybridisation of the embryos was performed, antibodies labeled and TUNEL performed. On another set of embryos, microangiography and imaging was performed and data quantified and analysed.

The present study searched for mutations responsible for causing ocular coloboma using a genetic screen. A mutant line designated vu270 was identified at 2 days post-fertilisation, where failure of the fusion of the optic fissure was evident. Two other phenotypes of the embryos were observed, being a larger head and no apparent red blood cells. The zebrafish injected with the mutation as stated earlier failed to generate red blood cells and thus, this study proved that the lmo2 gene has a crucial role for hematopoiesis. The study also condemns the zebrafish an appropriate model to study the roles of lmo2 in embryonic development.
In comparison to its role in red blood cell formation, the functions of lmo2 in vascular development are not as well known. Whilst previous studies demonstrated the requirement of lmo2 in angiogenesis<ref name=PMID11857074><pubmed>11857074</pubmed></ref>, the present study although reiterated this, showed abnormal formation of the blood vessels. This study further condemned the zebrafish as a relevant model to study the function of lmo2 in angiogenesis and the abnormalities associated with the vasculature that arises due to this gene, are a result of the genes’ role in maintaining vascular permeability and integrity. Therefore, it is evident that the absence of the lmo2 gene correlates with increased permeability of the vasculature.
Even more specifically, the results showed constrictions within the hyaloid artery and nerve, but severe dilatations in the hyaloid vein, indicating that lmo2 has different roles in the development of different blood vessels, or that in constricted vessels there is no flow. However, the idea that in constricted blood vessels there is no flow can be rid of as a conclusion since microangiography results showed blood flow.

In conclusion, the results of the present study demonstrate that abnormal blood vessels coursing through the optic fissure can indeed interfere with its closure.
[[Sensory - Vision Development]]

<references/>

==Online Assessment 10==

<pubmed>25085858</pubmed>

Due to the increased incidence and mortality rates of cardiovascular diseases such as ischaemic heart disease, aortic aneurysms and peripheral vascular diseases in Western society, such conditions have called for various treatment options, including vascular bypass grafting and replacements.<ref name=PMID16397155><pubmed>16397155</pubmed></ref> Much of the time, patients do not have suitable corresponding vessels for such bypass treatments or replacements and sometimes even result in thrombosis, infection and pseudoaneurysms.<ref name=PMID23197861><pubmed>23197861</pubmed></ref>This is of particular importance in the paediatric field, where children require multiple surgeries to accommodate for the lack of growth of the vasculature and synthetic vascular grafts. <ref name=PMID19099046><pubmed>19099046</pubmed></ref> Therefore, there is a great need for new and improved vascular grafts and vascular replacement treatment options and such a need has been the driving force for the development of tissue-engineered blood vessels. Pluripotent stem cells have developed into a promising source of cells due to their high proliferating capacity and high differentiation potential to form different types of cells. Previous research successfully differentiated embryonic stem cells and induced pluripotent stem cells from mice into smooth muscle cells, indicating their potential for use in regenerative medicine and vascular engineering.<ref name=PMID21439638><pubmed>21439638</pubmed></ref>
This study aims to create human induced pluripotent stem cells from patient primary aortic fibroblasts and turn them into functional smooth muscle cells. It also examines the ability of the derived smooth muscle cells to construct vascular tissues on predesigned three-dimensional biodegradable scaffolds.

The study isolates and cultures primary aortic fibroblasts from a heart transplant donor, by sterilizing the tissue, removing the tunica intima and separating the tunic media and adventitia. The tunica media and adventitia were then treated with various chemicals to wash out the fibroblasts and smooth muscle cells. Induced pluripotent stem cell lines were then generated and characterized, and then stem cells were then allowed to differentiate into smooth muscle cells. The muscle cells were then assayed based on their contractility and quantitative RT-PCR, flow cytometry, fabrications of 3D scaffolds, and construction of tissue-engineered vascular tissues was performed. Scanning electron microscopy, histological observations and statistical analyses were also conducted.

The study successfully isolated the fibroblasts from the primary aortic tissue and induced pluripotent stem cell lines could be maintained and expanded on MEF feeders and Matrigel-coated surfaces. Many of these stem cell lines maintained their pluripotency, evident by RT-PCR results showing SOX2, NANOG and OCT4 transcription factors. The study also found that using a smaller pore size within the macroporous scaffold was better able to support the smooth muscle cell proliferation, resulting in a higher smooth muscle cell density.

In conclusion, construction of a whole tissue-engineered blood vessel requires the addition of all the other different layers, and not just smooth muscles cells, for example, cells from the tunica intima, media and adventitia. Future studies will involve the culturing of these other cell types altogether and could have even greater implications for future regenerative medicine.

<references/>

User:Z3417753

2014-10-26T05:32:04Z

Z3417753:

{{StudentPage2014}}

==Lab Attendance==
* Lab 1 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 12:54, 6 August 2014 (EST)
* Lab 2 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:21, 13 August 2014 (EST)
* Lab 3 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 20 August 2014 (EST)
* Lab 4 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:45, 27 August 2014 (EST)
* Lab 5 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:48, 3 September 2014 (EST)
* Lab 6 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:50, 10 September 2014 (EST)
* Lab 7 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:31, 17 September 2014 (EST)
* Lab 8 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 24 September 2014 (EST)
* Lab 9 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:27, 8 October 2014 (EST)
* Lab 10 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:11, 15 October 2014 (EST)
* Lab 11 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:26, 22 October 2014 (EST)

==Online Assessment 1==

===Article 1===
<pubmed>23148203</pubmed>

This study is an analysis of the optimal time from oocyte to preimplantation embryo development for biopsy and preimplantation genetic screening. The discovery of the optimal time can then be used to detect any abnormal chromosomal separation patterns in embryos from older mothers (>40 years old). The study was a longitudinal cohort study involving 9 infertile couples and 21 sets of complete chromosomal screening data, including polar bodies 1 + 2 and their corresponding blastomeres and trophectoderm samples.

METHODS →infertile couples with a good response to controlled ovarian stimulation were enrolled in the study and underwent IVF. Polar bodies, blastomeres and trophectoderm samples were biopsied and analysed by array comparative genomic hybridisation. The chromosomal segregation patterns were analysed from these results and used to deduce the origin of aneuploidy. The results were also used to examine the accuracy of polar body and cleavage-stage preimplantation genetic screening strategies.

RESULTS → Since preimplantation genetic screening tests have been conducted at different times throughout the preimplantation window, it is possible that critical bits of information regarding chromosomal segregation patterns have been missed. Thus, by performing such tests at an optimal time, we are better able to understand these meiotic chromosomal segregation patterns and therefore potentially increase the success rates of in-vitro fertilisation. This study uses a sequential chromosome analysis of polar bodies and their corresponding embryos at both the cleavage and blastocyst stages in order to work out what stage is best to perform these genetic screening tests and biopsies, potentially increasing IVF success rate. The study showed that testing at the polar body stage was least accurate due to the high incidence of post-zygotic events and discovered that performing these tests later on in development (at the blastocyst stage) may produce more reliable results for the screenings, thereby achieving better chromosomal segregation pattern data. These results can now go on to be used for IVF research.

===Article 2===

<pubmed>23477909</pubmed>

This study examines the accuracy of using array comparative genomic hybridisation (array CGH) techniques for the analysis of first and second polar bodies in predicting aneuploidies of maternal meiotic origin in the cleavage stage embryos of women of advanced maternal age. It is known that aneuploidy is a common cause of pregnancy failure, miscarriage and abnormal pregnancy and most aneuploidy is due to maternal meiotic origin and increases exponentially as the mother approaches menopause.

METHOD → 20 couples requesting preimplantation genetic screening for advanced maternal age (=greater than or equal to 35 years old) and repeated implantation failure (more than 3 cycles), previous aneuploidy pregnancy or recurrent first trimester miscarriage underwent 16 controlled ovarian hyperstimulation cycles and 7 natural fresh cycles. Male partners had sperm parameters within the normal range except for 2 which had oligoasthenoteratozoospermia. Oocytes were retrieved by ultrasound-guided transvaginal aspiration 36 hours after beta-hCG administration. Once the oocytes were retrieved, biopsy of the first polar body was performed and the oocyte was inseminated using intracytoplasmic sperm injection. The following morning, each oocyte was checked for prouclei and extrusion of the second polar body to confirm fertilisation. The second polar body was then biopsied. The polar bodies were then analysed using array CGH analysis and the zona pellucida layer of the oocyte was dissolved. The zona-free embryo then underwent whole genome amplification and array CGH analysis in the cleavage stage.

RESULTS → It has been demonstrated in previous studies that a high correlation exists between the chromosomal status presented from polar body analysis and the actual chromosomes present in the zygotes of older mothers. Due to these results, this study uses polar body analysis and array CGH analysis of mature fertilised oocytes, to identify errors in meiosis within the polar bodies as well as the corresponding cleavage stage embryos. The results of the current study showed that nearly ALL aneuploidies detected in cleavage stage embryos were associated with copy number changes in the polar bodies (93%), indicating the high capability of polar bodies being used to predict aneuploidy and what is actually happening within the embryo.

--[[User:Z8600021|Mark Hill]] These are good articles and summaries. Reference link is formatted correctly (5/5)

==Online Assessment 2==
[[File:Fusion of two pairs of blastomeres inside 4-cell embryos.png|800px]]

Fusion of two pairs of blastomeres inside 4-cell embryos<ref><pubmed>23227157</pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515590/figure/pone-0050029-g003/]</ref>

<references/>

--[[User:Z8600021|Mark Hill]] Image appropriate for assessment and all associated information formatted correctly. You may want to include species (mouse) information with the image. (5/5)

==Online Assessment 3==
===Current Research Models and Findings===
--[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 22:57, 26 August 2014 (EST)

<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>

--[[User:Z8600021|Mark Hill]] These are relevant references, a slingle line describing why you have selected these would have been also useful to include. (4/5)

==Online Assessment 4==
===Cord Stem Cell Article Findings===

<pubmed>25101638</pubmed>

This article identifies acute liver failure as a devastating and debilitating illness that occurs within a short period of time, ultimately resulting in death of the patient if proper treatment is unavailable or it is simply too late to treat. It further identifies liver transplantation as the most effective treatment to date however, its application is limited due to an elevated risk of organ rejection and lack of liver donors. It is also known that human umbilical mesenchymal stem cells (hUCMSC) have the potential to differentiate into hepatocyte-like cells, functioning very similarly to hepatocytes as well as secrete certain factors to stimulate the proliferation of nearby hepatocytes, thereby promoting the rejuvenation of the host liver cells. The author hypothesised that by decreasing the amount of manipulation received by the mesenchymal stem cells in vitro, the carcinogenic risk was reduced. As a result, the therapeutic effect (amount of liver repair) of concurrently acting hUCMSC’s and hepatocyte-like cells can be ascertained by studying and comparing the two synchronous actions in acute liver failure mouse models.

The study induced acute liver failure in mouse models using D-galactosamine and lipopolysaccharide, causing the death of approximately 50% of the mice (necrosis of more than 50% of the hepatocytes). The mouse models’ therapeutic effects were then compared before and after the mesenchymal stem cells were differentiated into hepatocyte-like cells, by transplanting and injecting the cells into the tail vein. The results showed that almost ALL mouse were saved by the injection of the hepatocyte-like cells. Similarly, the injection of the hUCMSC’s also demonstrated their capability to repair liver damage, however, the population of these cells tested via the expression/ presence of human hepatocyte growth factor was minimal, suggesting that they allow the reversal of acute liver failure by differentiating into hepatocyte-like cells.

Overall, these results suggest that hUCMSC’s and hepatocyte-like cells are just as effective in therapeutic treatment of acute liver failure in mouse models and that hUCMSC’s play a larger role in stimulating the host hepatocyte repair.

===Vascular Shunts===

Three major vascular shunts exist within the circulatory system of the foetus:

1. FORAMEN OVALE --> the opening in the interatrial septum (wall between left and right atrium) that allows the flow of blood from the right atrium to the left atrium and has a valve to prevent backflow during the fetal period. It soon closes once right atrial pressure increases.
The foramen ovalis then becomes the FOSSA OVALIS postnatally.

2. DUCTUS ARTERIOSUS --> muscular vessel that connects the pulmonary trunk to the aorta, thereby diverting bloodflow to the lungs and going straight into the aorta. After birth, as the amount of oxygen increases, the smooth muscle in the walls constricts closing off the passage.
As the ductus arteriosus degenerates, all that is left behind if the LIGAMENTUM ARTERIOSUM.

3. DUCTUS VENOSUS --> a blood vessel that branches from the umbilical vein, allowing oxygenated blood from the placenta to be diverted from the fetal liver to the fetal heart.
This shunt closes slowly during infancy and degenerates into the LIGAMENTUM VENOSUM.

==Online Assessment 5==
Laryngeal-tracheo-oesophageal Cleft is a rare congenital anomaly where there is an abnormal posterior communication between the larynx and pharynx, extending down between the trachea and oesophagus.

Normally, the larynx develops simultaneously from the endoderm (arising from the foregut region) and the mesenchyme (arising from the 4th + 6th pharyngeal arches. The division of the foregut is due to the fusion of the lateral walls of the foregut in the region of the larynx, thereby forming a septum that divides the foregut into a central part = LARYNGEAL-TRACHEAL TUBE as well as a dorsal portion = OESOPHAGUS. The mesenchymal portion (= TRACHEAL-OESOPHAGEAL SEPTUM) is located between the digestive and respiratory tracts and is the result of the separation of the two tracts. Apoptotic epithelial cells are also present at this septum, mainly in the ventral portion, but inactive in the dorsal portion.

There are a few models that explain tracheal-oesophageal anomalies, including Laryngeal-tracheo-oesophageal Cleft:

• INTRAEMBRYONIC PRESSURE → an intense curvature of the cervical region during heart development places pressure upon the oesophagus and as a result displaces it, leading to growth abnormalities.

• EPITHELIAL OCCLUSION → the oesophagus is solid during a stage of development but it is soon recanalised. If the recanalisation does not occur, growth abnormalities may occur.

• VASCULAR OCCLUSION → an abnormally communicating vessel could lead to avascularisation in the foregut, resulting in abnormalities. In the case of Laryngeal-tracheo-oesophageal Cleft, this means the laryngeal region.

• DIFFERENTIAL CELL GROWTH → abnormal cell growth in the ventral or dorsal part of the developing trachea or oesophagus could result in defects of the two tracts.<ref><pubmed>22151899</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22151899]</ref>

<references/>

==Online Assessment 6==

===Research Article on Development of the Pancreas===
<pubmed>24375815</pubmed>

This article explores the role of pancreas-specific transcription factor 1a (PTF1a) in development of the pancreas, but makes a note of the difficulty associated with obtaining embryonic tissue specimens for experimentation. Due to the limitations associated with such embryonic material, embryonic stem cells (ESCs), which can be differentiated in vitro are used as a model system to study and examine the role of PTF1a in the development of the pancreas.
The study uses cell cultures, quantitative PCR, immunofluorescent staining, flow cytometry and western blot staining to demonstrate that PTF1a is required very early in development for arrangement of the pancreas from the foregut endoderm. The study shows that PTF1a drives differentiation of pancreatic cells from embryonic stem cells and this determines PTF1a to be an initiator of pancreatic differentiation in the form of ductal, endocrine and exocrine cells. The ectopic expression of PTF1a stimulated the ESCs to start differentiating into pancreas, causing the cells to activate PDX1 expression in bud-like structures that looked like early pancreas in vivo. The study also found that retinoic acid and nicotinamide signaling could regulate the ratio of endocrine to exocrine cell differentiation.
The future implications of this study may involve further studies utilizing the notion of the importance of activating PTF1a in the development of enhanced pancreatic differentiation for creating ESC-derived insulin (beta cells) expressing cells.

===Embryonic Layers and Tissues that Contribute to Tooth Development===

Teeth are part of the integumentary system and are formed by epithelial as well as mesenchymal interactions during development. They are largely formed by ECTODERM of the first pharyngeal arch, MESODERM and receive a major contribution from NEURAL CREST ECTOMESENCHYMAL cells. The neural crest mesenchymal cells change due to the enamel epithelium.

==Online Assessment 7==

'''TIME COURSE OF EMBRYONIC DEVELOPMENT OF THE HUMAN TESTIS'''

During fertilisation, genes determine the sexual fate of the organism and whether the organism is male or female is only revealed in fetal development when development of the external genitalia finally occurs. The presence of the Y chromosome leads to the development of testes in humans and their development is dependent on a single gene located on this Y chromosome known as Testis-determining factor (TDF).

When observing the morphology and cell biology of the developing testis, it is important to note that much of the research conducted on the subject involved the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy.

The next stage in embryonic development of the human testis is the formation of the genital ridge in an initial phase. This genital ridge is representative of the ambisexual stage in human embryos and is a bipotential gonad, meaning it possesses the capacity to differentiate into either female or male gonads. The next phase involves the development of a testis or ovary, which is entirely dependent upon the expression of the TDF gene SRY.

Initially, the gonads arise as paired structures within the intermediate mesoderm, where there are three parts that comprise the urogenital ridge: the pronephros (caudally), the mesonephros (central region where the gonad arises) and the metanephros (posteriorly, forming the kidney).
Cells that delaminate from the epithelium of the coelom provide a source of cells for the growing genital ridges and underlying cells from the mesonephros also expands the cell population in the gonadal primordia of males. Also, supporting cell precursors such as for Sertoli or Leydig cells are present within this early time period. The mesonephric ducts (Wolffian ducts) go on to form the ductal system of the male gonads and mesonephric tubules form shortly later, playing an important role in signaling surrounding areas for testis development.
Differentiation of testis occurs when the SRY gene is expressed within somatic cells, inducing them to form into Sertoli cells, which in turn, lead to the differentiation of all other cells present within the testis. Simultaneously, the gonad increases its size due to increased growth and movement of cells from the adjacent mesonephros. These cells give rise to peritubular myoid cells, endothelial cells that go on to form vasculature of the male gonad and to Leydig cells. The next stages involve testis-cord formation, Leydig cell formation (which secretes androgens required for fetal masculinization and the development of external genitalia). <ref><pubmed>17237341 </pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/17237341]</ref>

<references/>

'''Image: Diagram representing certain persistent portions of the mesonephros in the male'''
[[File:Bailey309.jpg|500px]]

==Online Assessment 8==
===Peer Reviews===
====Group Project 1====

''Respiratory Development''

The introduction is very informative and I particularly like how it describes the embryonic development of the respiratory system as well, since in order to understand what is happening in the fetal period, it is important to first understand what happened before that in the embryonic period. Perhaps the introduction could also introduce what information the page is going to contain.

The timeline is well presented in a table form, however maybe it would be better suited to be in the introduction section. The table could also incorporate the use of histological images to illustrate the differences between the time periods. Also, the sub sections titled ‘current models’ and ‘current research and findings’ could be part of a larger section and not fall under the ‘Lung Development Stages’ section.

There is no information as yet under ‘Current models’ however extensive research seems to be conducted on ‘current research findings’. Perhaps it would be better to include more journal articles in this section. The use of dot points and numbering systems is also very effective in allowing the information to be easily read and flow. More articles also need to be covered in the ‘Historic findings section’ as it is very brief at the moment with only a few sentences on each article.

The ‘abnormalities’ section is very well done with an abundance of conditions however more images should be uploaded for each abnormality in order to see what it visually presents as in the fetus and also to make the page look nicer.

The images uploaded onto the page contain adequate information explaining them, copyright information as well as the student image template, which is good. There is one student drawn image, which is also great, but maybe some more would further illustrate the group’s understanding of their topic.

The referencing is done correctly mostly throughout the page but is scattered throughout every section so perhaps it would be better to have them in one section at the bottom of the page under the heading entitled ‘References’ and numbered as they appear in the text. In-text citations are throughout and appear to be done correctly.

Overall, this is a very good effort and a bit of editing will make the page look much more neater and organized. Keep up the great work!

====Group Project 2====

''Renal Development''

The introduction provides a very informative description of the functions of the kidney and bladder. Perhaps it would be good to give some more details of the embryonic development just to quickly summarise what has been happening with the fetus up until this point. Also, maybe the introduction should introduce what the page’s content is going to cover. The order of historic findings and then developmental timeline is appropriate as historic findings can be used to compile the timeline. It would also be useful to have the timeline in a table format to make the page look neater and more simplified. Also, there is no research done on ‘historic findings’ so need to address that before final submission.

‘Current research models’ section is good but brief and requires more extensive research as only two articles are cited. There should be information on current models used to study renal development as well as current research and findings. The image in this section is well presented, with appropriate titling, referencing, image descriptions and copyright information with the student image template.
Sections 1.5-1.8 should be smaller sub headings under the larger heading ‘System Development’ and perhaps should go at the top of the page, beneath the introduction seeing as in order to understand research and historic findings, it is necessary to understand renal development first.

It is very good that there is a small section on early development, however maybe it would be better to have it more briefly explained, perhaps in the form of a student drawn diagram or presented as a table. There also is a problem with the image uploaded in the early development section, so should fix that before final submission. The ‘abnormalities’ section is also done well however more conditions should be listed and described with pictures for each one. There are also only abnormalities of the kidneys listed, so maybe it would be better to have more of the other components of the renal system as well (bladder, ureter, urethra).

Also, maybe more information regarding the anatomy of the kidneys and renal system should be added, as this is an anatomy course. Some images are also missing the student image template.
Most images are uploaded correctly with the right information, maybe more would make the page look more aesthetically pleasing as well as assist learning.

Referencing is done correctly with a numbering system and in-text citations are also correct. The in-text referencing in the ‘anatomical position’ sub section of ‘fetal development’ of the ‘Kidney’ section is not referenced appropriately so just fix that minor problem.

Overall, this is great work and should just include more information in certain sections and upload more images, preferably some student drawn images as well. Well done!

====Group Project 3====

''Gastrointestinal Development''

‘GIT system overview’ section is good but requires more information to introduce the GIT and what the page is going to have information on. Timeline could form part of this section and could also preferable be in the form of a student drawn image or even a table. The overview section also contains no in-text citations. It’s a great idea to split the GIT into the three parts: foregut, midgut and hindgut to aid in understanding. There is not much information on recent findings without any mention of current models as well so perhaps it would be best to address this before final submission.

In the foregut section there is not much mention of blood supply or innervation as was done for midgut and hindgut. Student drawn images are very impressive and referenced correctly with the student template, description, title and copyright information. The features of the midgut section could include some histological drawings or images. The ‘abnormalities’ section does not contain many in-text citations in one of the paragraphs and could include more deformities listed and described with more images, as well as information on how to treat and manage such disorders later in life. There is also no information or images addressing historical findings or current models so this needs to be looked into.

The references are correctly done and ordered, and are present at the bottom of the page. Some of the in-text citations aren’t throughout the text like they should be, for example, in the stomach, liver and gallbladder, and oesophagus sections.

Overall, good effort so far but more extensive research needs to be conducted for models and findings and more information for Abnormalities, as well as a few minor edits to make the page present more nicely.

====Group Project 5====

''Integumentary Development''

This page looks very neat and well organised, with an introduction that explains exactly what is going to appear and be discussed on the page. The Development Overview section is very well done, with the appropriate use of subheadings and content. The use of dot points is very effective, making the page look neater. Perhaps it would be good to draw a histological diagram of the skin layers, and uploading it to the skin development section. Specialised cells or important names throughout the page could be highlighted in bold or underlined as well, to highlight important terms and make it easier to learn and remember from. The title ‘Some Recent Findings’ accurately portrays what we as students can only do, which is identify SOME of the recent findings. This section could have more than 2 recent findings however and could be further subdivided by subheadings into the different components of the integumentary system – perhaps have 2-3 research articles for each component of the system. Historic findings are well researched but some more information would be good. The ‘Abnormalities’ section is so far the best looking section as it seems it is almost completed. Perhaps a few more abnormalities would be even better.

The table of the timeline in the ‘Development Overview’ section is superbly done and the use of histological images is fantastic as it provides the anatomical information visually. When I clicked on an image however, there was no proper referencing of the image and the copyright information and student image information was not present. The images are described very well.
One image has a problem and is present in red writing, so might need to remove this as something is wrong with the file and it could not be uploaded. There are no student-drawn images and I think if this group did this, it would really benefit their project and emphasise their understanding.

The ‘Some Recent Findings’ section has a purple background, which makes the page look more aesthetically pleasing and less monochrome. I like the ‘More recent papers’ box that can be expanded to reveal any more research papers related to the integumentary fetal development, in case anyone wants to have a further read- very clever.

Journal articles are correctly referenced but website references need to be improved upon- to find how to do this go to the ‘How to reference’ page. References are all over the place and need to be compiled under each heading or one main heading titled ‘References’ at the bottom of the page.

Overall, this page is looking fantastic at this point in time so keep up the great work!

====Group Project 6====

''Endocrine Development''

An introduction could be very useful to summarise what the page is going to discuss. Sections 1.2-1.11 could all be subheadings under the main heading ‘System Development’, and then each of these subheading could be further divided into smaller subheadings with timeline, introduction detailing structure/ function of the endocrine organ. It is however very well done how the headings of each organ are then further subdivided into ‘abnormalities’, ‘research findings’ and ‘timeline’. However, the fact that each section has its own references and is subdivided as such, shows that even though the page may appear more ordered, there appears to be little communication between group members at this stage. So perhaps a goal could be to make the page look like one flowing work piece as opposed to sections that each person has done.

I think the content is very well researched and I like the way each organ of the endocrine system is discussed, as all are important in fetal development. The use of images is appropriate and very well done as they are referenced correctly and when you click on an image it takes you to a new page showing the student image template, copyright information as well as extra information regarding the image. There are no student-drawn images however, so perhaps it could be possible to draw a flow chart perhaps of gonadal fetal development. The use of tables is also done very well and is frequent throughout the page, with some being used to illustrate the anatomical development of certain organs, for example, the adrenal gland and pancreas. The graphs are also useful in portraying information from research findings.

The project page is missing information regarding historic findings, and I think that if this page is going to have a main heading for Abnormalities, then the group should put all their information regarding abnormalities under this section. Although it is not an endocrine organ that grows within the developing foetus, but is an important part of the mother, there is not much information on the page regarding the placenta. This section needs to be completed as the placenta is an important source of hormones and acts as an endocrine organ during the pregnancy, sustaining the foetus.

It is good that there are many references, indicating thorough research into the endocrine system with each organ heading have its own sources, however I think these references need to be ordered better. The actual referencing is done correctly, however in-text referencing is absent, so it may be best to fix this. Most images are referenced correctly as well.

Overall, keep up the good work, but just edit the page to make it look neater and finish the sections you need to.

====Group Project 7====

''Neural (CNS) Development''

This project page is very nicely organised with the group clearly specifying what aspect of neural development they are covering, being the CNS. The use of headings and subheadings is done very neatly, however sections 1.1-1.5 could be subheadings for the larger title ‘system development’. The key points have been clearly described but there is no referencing throughout the ‘Introduction’, ‘Brain development’ and ‘Abnormalities’ sections. Most key points have at least some information on them which is good for this stage of the project; however some of the headings without could use some more work.

The choice of content is highly appropriate and the use of diagrams and pictures help show the groups understanding of the project thus far. I particularly like the use of subheadings in this project as they make the page look neater and organised. The image showing the timeline of fetal neural development is good however perhaps it would be better to draw or make a timeline on the computer in order to show better understanding of the time course of fetal development. Most images that have been uploaded are also well referenced and when clicking onto them, it takes the reader to a page that has more information related to the image. The table to describe anatomical details is also done well and is important that such a key point is mentioned seeing as this is an anatomy course.

I also really like how the ‘Current research, models and findings’ section is split into ‘Current research’ and ‘Future Research’, however it seems future research needs to be further looked into. The ‘Abnormalities’ section is done very well, with multiple abnormalities listed with images used to show each one. The bolding of several key words is seen and is helpful in showing understanding of some of the key points. There are also no historic findings so try and find some information on that.

Referencing is correctly done with most references being in one main section at the end, and ordered correctly. In-cite referencing is also done correctly. All images are correctly referenced with copyright information present and the student image template. I also like the way the current research findings sources have been referenced with the use of dot points assisting learning by not just presenting to the reader as a blob of information.

Overall, well done group 7! Keep up the great work!

====Group Project 8====

''Musculoskeletal Development''

The key points of musculoskeletal development appear as headings however there is still much that needs to be clearly discussed beneath each of these points. The main headings are good and specific but some are way too specific and should be under much larger headings, for example, 1.2-1.9 could be subheadings that come under the heading ‘System Development’. ‘Background embryonic development’ is useful to understand but perhaps it is better to not have so much detail, or summarise it in a table. The ‘Abnormalities’ heading is done well, with one disease listed (Duchenne Muscular Dystrophy). It might be better to have more than one abnormality listed and clearly described as well. I particularly like the use of statistics and genetic references. It seems most of the key points relating to system development have been clearly described, but some tidying up in terms of editing needs to be done.

Also, more work needs to be done on historic findings, current research, models and findings. Once all the research parts are completed, the timeline can be correctly constructed. Also like the idea of putting a timeline and the heading shows that this is intended. More subheadings could be used to make the page look more organised and pleasing to the eye.

There are also no graphs or tables as well as pictures. A table could be used to make the timeline or highlight the differences between the second, third trimesters and neonatal periods of fetal muscular development. Maybe the initial heading of the page should be changed to ‘Muscular Fetal Development’ to indicate that muscular development is actually being covered instead of both muscular and skeletal. There also isn’t much information regarding limb fetal development, so maybe it would be good to go through that on a deeper level.

It could also help to have images loaded onto the page or to draw flow diagrams to assist in the description of how the muscles develop in the fetal period. For example, upload an image showing the difference between slow twitch and fast twitch muscle fibres or draw a flow chart to show better understanding of the molecular and cellular regulation of fetal myogenesis.

References need to be in one larger section at the end under the heading ‘References’, not two and scattered throughout as is seen. The major section of references appears to be referenced correctly and in-cite references are done very well. There are also many references which are good and show that this group has thoroughly researched their topic.

Overall, this group has done very well and just needs to add more information for certain headings, as well as organise the page a bit better in neater headings and subheadings. Pictures should be added, as well as graphs, tables and own student-drawn images.

==Online Assessment 9==

'''Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos'''.<ref name=PMID22819672><pubmed>22819672</pubmed></ref>

One of the early stages of embryonic eye development involves the invagination of the optic vesicle resulting in the formation of the bilayered optic cup with a groove on its anterior aspect. This groove is termed the optic fissure (also known as embryonic fissure) and creates an opening into which the hyaloid artery and vein can enter and exit the developing eye. As time passes, this fissure begins to fuse back together, enclosing the hyaloid vessels and this event occurs between the 6th to 7th weeks of gestation<ref name=PMID1628748><pubmed>1628748</pubmed></ref>. The zebrafish is a suitable model for observing such development due to its faster time course, so in embryos the fissure takes approximately 2 days post-fertilisation to close. When this optic fissure fails to close, a disorder known as ocular coloboma occurs, leading to impaired vision and possibly blindness later on in life<ref name=PMID16932062><pubmed>16932062</pubmed></ref>.

Further studies conducted on zebrafish and mouse models have shown genetic mutations are responsible for the abnormal patterning of the optic vesicle and decreased gene expression involving the anterior eye and periocular mesenchyme and subsequently, excess tissue cell proliferation and fusion abnormalities<ref name=PMID17609112><pubmed>17609112</pubmed></ref>. Therefore, it is evident that there are many mechanisms responsible for the fusion event of the optic fissure but no studies have previously attempted to understand the mechanisms that result in the failure of this fusion event. Hence, the present study hypothesizes that the hyaloid vasculature is somehow related to this fusion event and if there are variations in the blood vessels such as dilatations, then the optic fissure does not close properly.

The study uses zebrafish Imo2 mutants that fail to close the optic fissure at 2 days post-fertilisation. This was done by isolating RNA from Imo2 mutants (1-day post-fertilisation) and cloning it using vectors. The gene mutation was then introduced into the RNA and injected into the embryo. Once the 2-day post-fertilisation window passed, embryos were fixed and tissues were sectioned and stained. In-situ hybridisation of the embryos was performed, antibodies labeled and TUNEL performed. On another set of embryos, microangiography and imaging was performed and data quantified and analysed.

The present study searched for mutations responsible for causing ocular coloboma using a genetic screen. A mutant line designated vu270 was identified at 2 days post-fertilisation, where failure of the fusion of the optic fissure was evident. Two other phenotypes of the embryos were observed, being a larger head and no apparent red blood cells. The zebrafish injected with the mutation as stated earlier failed to generate red blood cells and thus, this study proved that the lmo2 gene has a crucial role for hematopoiesis. The study also condemns the zebrafish an appropriate model to study the roles of lmo2 in embryonic development.
In comparison to its role in red blood cell formation, the functions of lmo2 in vascular development are not as well known. Whilst previous studies demonstrated the requirement of lmo2 in angiogenesis<ref name=PMID11857074><pubmed>11857074</pubmed></ref>, the present study although reiterated this, showed abnormal formation of the blood vessels. This study further condemned the zebrafish as a relevant model to study the function of lmo2 in angiogenesis and the abnormalities associated with the vasculature that arises due to this gene, are a result of the genes’ role in maintaining vascular permeability and integrity. Therefore, it is evident that the absence of the lmo2 gene correlates with increased permeability of the vasculature.
Even more specifically, the results showed constrictions within the hyaloid artery and nerve, but severe dilatations in the hyaloid vein, indicating that lmo2 has different roles in the development of different blood vessels, or that in constricted vessels there is no flow. However, the idea that in constricted blood vessels there is no flow can be rid of as a conclusion since microangiography results showed blood flow.

In conclusion, the results of the present study demonstrate that abnormal blood vessels coursing through the optic fissure can indeed interfere with its closure.
[[Sensory - Vision Development]]

<references/>

==Online Assessment 10==

<pubmed>25085858</pubmed>

Due to the increased incidence and mortality rates of cardiovascular diseases such as ischaemic heart disease, aortic aneurysms and peripheral vascular diseases in Western society, such conditions have called for various treatment options, including vascular bypass grafting and replacements.<ref name=PMID16397155><pubmed>16397155</pubmed></ref> Much of the time, patients do not have suitable corresponding vessels for such bypass treatments or replacements and sometimes even result in thrombosis, infection and pseudoaneurysms.<ref name=PMID23197861><pubmed>23197861</pubmed></ref>This is of particular importance in the paediatric field, where children require multiple surgeries to accommodate for the lack of growth of the vasculature and synthetic vascular grafts. <ref name=PMID19099046><pubmed>19099046</pubmed></ref> Therefore, there is a great need for new and improved vascular grafts and vascular replacement treatment options and such a need has been the driving force for the development of tissue-engineered blood vessels. Pluripotent stem cells have developed into a promising source of cells due to their high proliferating capacity and high differentiation potential to form different types of cells. Previous research successfully differentiated embryonic stem cells and induced pluripotent stem cells from mice into smooth muscle cells, indicating their potential for use in regenerative medicine and vascular engineering.<ref name=PMID21439638><pubmed>21439638</pubmed></ref>
This study aims to create human induced pluripotent stem cells from patient primary aortic fibroblasts and turn them into functional smooth muscle cells. It also examines the ability of the derived smooth muscle cells to construct vascular tissues on predesigned three-dimensional biodegradable scaffolds.

The study isolates and cultures primary aortic fibroblasts from a heart transplant donor, by sterilizing the tissue, removing the tunica intima and separating the tunic media and adventitia. The tunica media and adventitia were then treated with various chemicals to wash out the fibroblasts and smooth muscle cells. Induced pluripotent stem cell lines were then generated and characterized, and then stem cells were then allowed to differentiate into smooth muscle cells. The muscle cells were then assayed based on their contractility and quantitative RT-PCR, flow cytometry, fabrications of 3D scaffolds, and construction of tissue-engineered vascular tissues was performed. Scanning electron microscopy, histological observations and statistical analyses were also conducted.

The study successfully isolated the fibroblasts from the primary aortic tissue and induced pluripotent stem cell lines could be maintained and expanded on MEF feeders and Matrigel-coated surfaces. Many of these stem cell lines maintained their pluripotency, evident by RT-PCR results showing SOX2, NANOG and OCT4 transcription factors. The study also found that using a smaller pore size within the macroporous scaffold was better able to support the smooth muscle cell proliferation, resulting in a higher smooth muscle cell density.

In conclusion, construction of a whole tissue-engineered blood vessel requires the addition of all the other different layers, and not just smooth muscles cells, for example, cells from the tunica intima, media and adventitia. Future studies will involve the culturing of these other cell types altogether and could have even greater implications for future regenerative medicine.

User:Z3417753

2014-10-26T04:32:35Z

Z3417753:

{{StudentPage2014}}

==Lab Attendance==
* Lab 1 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 12:54, 6 August 2014 (EST)
* Lab 2 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:21, 13 August 2014 (EST)
* Lab 3 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 20 August 2014 (EST)
* Lab 4 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:45, 27 August 2014 (EST)
* Lab 5 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:48, 3 September 2014 (EST)
* Lab 6 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:50, 10 September 2014 (EST)
* Lab 7 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:31, 17 September 2014 (EST)
* Lab 8 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:37, 24 September 2014 (EST)
* Lab 9 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:27, 8 October 2014 (EST)
* Lab 10 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:11, 15 October 2014 (EST)
* Lab 11 --[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 11:26, 22 October 2014 (EST)

==Online Assessment 1==

===Article 1===
<pubmed>23148203</pubmed>

This study is an analysis of the optimal time from oocyte to preimplantation embryo development for biopsy and preimplantation genetic screening. The discovery of the optimal time can then be used to detect any abnormal chromosomal separation patterns in embryos from older mothers (>40 years old). The study was a longitudinal cohort study involving 9 infertile couples and 21 sets of complete chromosomal screening data, including polar bodies 1 + 2 and their corresponding blastomeres and trophectoderm samples.

METHODS →infertile couples with a good response to controlled ovarian stimulation were enrolled in the study and underwent IVF. Polar bodies, blastomeres and trophectoderm samples were biopsied and analysed by array comparative genomic hybridisation. The chromosomal segregation patterns were analysed from these results and used to deduce the origin of aneuploidy. The results were also used to examine the accuracy of polar body and cleavage-stage preimplantation genetic screening strategies.

RESULTS → Since preimplantation genetic screening tests have been conducted at different times throughout the preimplantation window, it is possible that critical bits of information regarding chromosomal segregation patterns have been missed. Thus, by performing such tests at an optimal time, we are better able to understand these meiotic chromosomal segregation patterns and therefore potentially increase the success rates of in-vitro fertilisation. This study uses a sequential chromosome analysis of polar bodies and their corresponding embryos at both the cleavage and blastocyst stages in order to work out what stage is best to perform these genetic screening tests and biopsies, potentially increasing IVF success rate. The study showed that testing at the polar body stage was least accurate due to the high incidence of post-zygotic events and discovered that performing these tests later on in development (at the blastocyst stage) may produce more reliable results for the screenings, thereby achieving better chromosomal segregation pattern data. These results can now go on to be used for IVF research.

===Article 2===

<pubmed>23477909</pubmed>

This study examines the accuracy of using array comparative genomic hybridisation (array CGH) techniques for the analysis of first and second polar bodies in predicting aneuploidies of maternal meiotic origin in the cleavage stage embryos of women of advanced maternal age. It is known that aneuploidy is a common cause of pregnancy failure, miscarriage and abnormal pregnancy and most aneuploidy is due to maternal meiotic origin and increases exponentially as the mother approaches menopause.

METHOD → 20 couples requesting preimplantation genetic screening for advanced maternal age (=greater than or equal to 35 years old) and repeated implantation failure (more than 3 cycles), previous aneuploidy pregnancy or recurrent first trimester miscarriage underwent 16 controlled ovarian hyperstimulation cycles and 7 natural fresh cycles. Male partners had sperm parameters within the normal range except for 2 which had oligoasthenoteratozoospermia. Oocytes were retrieved by ultrasound-guided transvaginal aspiration 36 hours after beta-hCG administration. Once the oocytes were retrieved, biopsy of the first polar body was performed and the oocyte was inseminated using intracytoplasmic sperm injection. The following morning, each oocyte was checked for prouclei and extrusion of the second polar body to confirm fertilisation. The second polar body was then biopsied. The polar bodies were then analysed using array CGH analysis and the zona pellucida layer of the oocyte was dissolved. The zona-free embryo then underwent whole genome amplification and array CGH analysis in the cleavage stage.

RESULTS → It has been demonstrated in previous studies that a high correlation exists between the chromosomal status presented from polar body analysis and the actual chromosomes present in the zygotes of older mothers. Due to these results, this study uses polar body analysis and array CGH analysis of mature fertilised oocytes, to identify errors in meiosis within the polar bodies as well as the corresponding cleavage stage embryos. The results of the current study showed that nearly ALL aneuploidies detected in cleavage stage embryos were associated with copy number changes in the polar bodies (93%), indicating the high capability of polar bodies being used to predict aneuploidy and what is actually happening within the embryo.

--[[User:Z8600021|Mark Hill]] These are good articles and summaries. Reference link is formatted correctly (5/5)

==Online Assessment 2==
[[File:Fusion of two pairs of blastomeres inside 4-cell embryos.png|800px]]

Fusion of two pairs of blastomeres inside 4-cell embryos<ref><pubmed>23227157</pubmed>| [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515590/figure/pone-0050029-g003/]</ref>

<references/>

--[[User:Z8600021|Mark Hill]] Image appropriate for assessment and all associated information formatted correctly. You may want to include species (mouse) information with the image. (5/5)

==Online Assessment 3==
===Current Research Models and Findings===
--[[User:Z3417753|Z3417753]] ([[User talk:Z3417753|talk]]) 22:57, 26 August 2014 (EST)

<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>

--[[User:Z8600021|Mark Hill]] These are relevant references, a slingle line describing why you have selected these would have been also useful to include. (4/5)

==Online Assessment 4==
===Cord Stem Cell Article Findings===

<pubmed>25101638</pubmed>

This article identifies acute liver failure as a devastating and debilitating illness that occurs within a short period of time, ultimately resulting in death of the patient if proper treatment is unavailable or it is simply too late to treat. It further identifies liver transplantation as the most effective treatment to date however, its application is limited due to an elevated risk of organ rejection and lack of liver donors. It is also known that human umbilical mesenchymal stem cells (hUCMSC) have the potential to differentiate into hepatocyte-like cells, functioning very similarly to hepatocytes as well as secrete certain factors to stimulate the proliferation of nearby hepatocytes, thereby promoting the rejuvenation of the host liver cells. The author hypothesised that by decreasing the amount of manipulation received by the mesenchymal stem cells in vitro, the carcinogenic risk was reduced. As a result, the therapeutic effect (amount of liver repair) of concurrently acting hUCMSC’s and hepatocyte-like cells can be ascertained by studying and comparing the two synchronous actions in acute liver failure mouse models.

The study induced acute liver failure in mouse models using D-galactosamine and lipopolysaccharide, causing the death of approximately 50% of the mice (necrosis of more than 50% of the hepatocytes). The mouse models’ therapeutic effects were then compared before and after the mesenchymal stem cells were differentiated into hepatocyte-like cells, by transplanting and injecting the cells into the tail vein. The results showed that almost ALL mouse were saved by the injection of the hepatocyte-like cells. Similarly, the injection of the hUCMSC’s also demonstrated their capability to repair liver damage, however, the population of these cells tested via the expression/ presence of human hepatocyte growth factor was minimal, suggesting that they allow the reversal of acute liver failure by differentiating into hepatocyte-like cells.

Overall, these results suggest that hUCMSC’s and hepatocyte-like cells are just as effective in therapeutic treatment of acute liver failure in mouse models and that hUCMSC’s play a larger role in stimulating the host hepatocyte repair.

===Vascular Shunts===

Three major vascular shunts exist within the circulatory system of the foetus:

1. FORAMEN OVALE --> the opening in the interatrial septum (wall between left and right atrium) that allows the flow of blood from the right atrium to the left atrium and has a valve to prevent backflow during the fetal period. It soon closes once right atrial pressure increases.
The foramen ovalis then becomes the FOSSA OVALIS postnatally.

2. DUCTUS ARTERIOSUS --> muscular vessel that connects the pulmonary trunk to the aorta, thereby diverting bloodflow to the lungs and going straight into the aorta. After birth, as the amount of oxygen increases, the smooth muscle in the walls constricts closing off the passage.
As the ductus arteriosus degenerates, all that is left behind if the LIGAMENTUM ARTERIOSUM.

3. DUCTUS VENOSUS --> a blood vessel that branches from the umbilical vein, allowing oxygenated blood from the placenta to be diverted from the fetal liver to the fetal heart.
This shunt closes slowly during infancy and degenerates into the LIGAMENTUM VENOSUM.

==Online Assessment 5==
Laryngeal-tracheo-oesophageal Cleft is a rare congenital anomaly where there is an abnormal posterior communication between the larynx and pharynx, extending down between the trachea and oesophagus.

Normally, the larynx develops simultaneously from the endoderm (arising from the foregut region) and the mesenchyme (arising from the 4th + 6th pharyngeal arches. The division of the foregut is due to the fusion of the lateral walls of the foregut in the region of the larynx, thereby forming a septum that divides the foregut into a central part = LARYNGEAL-TRACHEAL TUBE as well as a dorsal portion = OESOPHAGUS. The mesenchymal portion (= TRACHEAL-OESOPHAGEAL SEPTUM) is located between the digestive and respiratory tracts and is the result of the separation of the two tracts. Apoptotic epithelial cells are also present at this septum, mainly in the ventral portion, but inactive in the dorsal portion.

There are a few models that explain tracheal-oesophageal anomalies, including Laryngeal-tracheo-oesophageal Cleft:

• INTRAEMBRYONIC PRESSURE → an intense curvature of the cervical region during heart development places pressure upon the oesophagus and as a result displaces it, leading to growth abnormalities.

• EPITHELIAL OCCLUSION → the oesophagus is solid during a stage of development but it is soon recanalised. If the recanalisation does not occur, growth abnormalities may occur.

• VASCULAR OCCLUSION → an abnormally communicating vessel could lead to avascularisation in the foregut, resulting in abnormalities. In the case of Laryngeal-tracheo-oesophageal Cleft, this means the laryngeal region.

• DIFFERENTIAL CELL GROWTH → abnormal cell growth in the ventral or dorsal part of the developing trachea or oesophagus could result in defects of the two tracts.<ref><pubmed>22151899</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22151899]</ref>

<references/>

==Online Assessment 6==

===Research Article on Development of the Pancreas===
<pubmed>24375815</pubmed>

This article explores the role of pancreas-specific transcription factor 1a (PTF1a) in development of the pancreas, but makes a note of the difficulty associated with obtaining embryonic tissue specimens for experimentation. Due to the limitations associated with such embryonic material, embryonic stem cells (ESCs), which can be differentiated in vitro are used as a model system to study and examine the role of PTF1a in the development of the pancreas.
The study uses cell cultures, quantitative PCR, immunofluorescent staining, flow cytometry and western blot staining to demonstrate that PTF1a is required very early in development for arrangement of the pancreas from the foregut endoderm. The study shows that PTF1a drives differentiation of pancreatic cells from embryonic stem cells and this determines PTF1a to be an initiator of pancreatic differentiation in the form of ductal, endocrine and exocrine cells. The ectopic expression of PTF1a stimulated the ESCs to start differentiating into pancreas, causing the cells to activate PDX1 expression in bud-like structures that looked like early pancreas in vivo. The study also found that retinoic acid and nicotinamide signaling could regulate the ratio of endocrine to exocrine cell differentiation.
The future implications of this study may involve further studies utilizing the notion of the importance of activating PTF1a in the development of enhanced pancreatic differentiation for creating ESC-derived insulin (beta cells) expressing cells.

===Embryonic Layers and Tissues that Contribute to Tooth Development===

Teeth are part of the integumentary system and are formed by epithelial as well as mesenchymal interactions during development. They are largely formed by ECTODERM of the first pharyngeal arch, MESODERM and receive a major contribution from NEURAL CREST ECTOMESENCHYMAL cells. The neural crest mesenchymal cells change due to the enamel epithelium.

==Online Assessment 7==

'''TIME COURSE OF EMBRYONIC DEVELOPMENT OF THE HUMAN TESTIS'''

During fertilisation, genes determine the sexual fate of the organism and whether the organism is male or female is only revealed in fetal development when development of the external genitalia finally occurs. The presence of the Y chromosome leads to the development of testes in humans and their development is dependent on a single gene located on this Y chromosome known as Testis-determining factor (TDF).

When observing the morphology and cell biology of the developing testis, it is important to note that much of the research conducted on the subject involved the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy.

The next stage in embryonic development of the human testis is the formation of the genital ridge in an initial phase. This genital ridge is representative of the ambisexual stage in human embryos and is a bipotential gonad, meaning it possesses the capacity to differentiate into either female or male gonads. The next phase involves the development of a testis or ovary, which is entirely dependent upon the expression of the TDF gene SRY.

Initially, the gonads arise as paired structures within the intermediate mesoderm, where there are three parts that comprise the urogenital ridge: the pronephros (caudally), the mesonephros (central region where the gonad arises) and the metanephros (posteriorly, forming the kidney).
Cells that delaminate from the epithelium of the coelom provide a source of cells for the growing genital ridges and underlying cells from the mesonephros also expands the cell population in the gonadal primordia of males. Also, supporting cell precursors such as for Sertoli or Leydig cells are present within this early time period. The mesonephric ducts (Wolffian ducts) go on to form the ductal system of the male gonads and mesonephric tubules form shortly later, playing an important role in signaling surrounding areas for testis development.
Differentiation of testis occurs when the SRY gene is expressed within somatic cells, inducing them to form into Sertoli cells, which in turn, lead to the differentiation of all other cells present within the testis. Simultaneously, the gonad increases its size due to increased growth and movement of cells from the adjacent mesonephros. These cells give rise to peritubular myoid cells, endothelial cells that go on to form vasculature of the male gonad and to Leydig cells. The next stages involve testis-cord formation, Leydig cell formation (which secretes androgens required for fetal masculinization and the development of external genitalia). <ref><pubmed>17237341 </pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/17237341]</ref>

<references/>

'''Image: Diagram representing certain persistent portions of the mesonephros in the male'''
[[File:Bailey309.jpg|500px]]

==Online Assessment 8==
===Peer Reviews===
====Group Project 1====

''Respiratory Development''

The introduction is very informative and I particularly like how it describes the embryonic development of the respiratory system as well, since in order to understand what is happening in the fetal period, it is important to first understand what happened before that in the embryonic period. Perhaps the introduction could also introduce what information the page is going to contain.

The timeline is well presented in a table form, however maybe it would be better suited to be in the introduction section. The table could also incorporate the use of histological images to illustrate the differences between the time periods. Also, the sub sections titled ‘current models’ and ‘current research and findings’ could be part of a larger section and not fall under the ‘Lung Development Stages’ section.

There is no information as yet under ‘Current models’ however extensive research seems to be conducted on ‘current research findings’. Perhaps it would be better to include more journal articles in this section. The use of dot points and numbering systems is also very effective in allowing the information to be easily read and flow. More articles also need to be covered in the ‘Historic findings section’ as it is very brief at the moment with only a few sentences on each article.

The ‘abnormalities’ section is very well done with an abundance of conditions however more images should be uploaded for each abnormality in order to see what it visually presents as in the fetus and also to make the page look nicer.

The images uploaded onto the page contain adequate information explaining them, copyright information as well as the student image template, which is good. There is one student drawn image, which is also great, but maybe some more would further illustrate the group’s understanding of their topic.

The referencing is done correctly mostly throughout the page but is scattered throughout every section so perhaps it would be better to have them in one section at the bottom of the page under the heading entitled ‘References’ and numbered as they appear in the text. In-text citations are throughout and appear to be done correctly.

Overall, this is a very good effort and a bit of editing will make the page look much more neater and organized. Keep up the great work!

====Group Project 2====

''Renal Development''

The introduction provides a very informative description of the functions of the kidney and bladder. Perhaps it would be good to give some more details of the embryonic development just to quickly summarise what has been happening with the fetus up until this point. Also, maybe the introduction should introduce what the page’s content is going to cover. The order of historic findings and then developmental timeline is appropriate as historic findings can be used to compile the timeline. It would also be useful to have the timeline in a table format to make the page look neater and more simplified. Also, there is no research done on ‘historic findings’ so need to address that before final submission.

‘Current research models’ section is good but brief and requires more extensive research as only two articles are cited. There should be information on current models used to study renal development as well as current research and findings. The image in this section is well presented, with appropriate titling, referencing, image descriptions and copyright information with the student image template.
Sections 1.5-1.8 should be smaller sub headings under the larger heading ‘System Development’ and perhaps should go at the top of the page, beneath the introduction seeing as in order to understand research and historic findings, it is necessary to understand renal development first.

It is very good that there is a small section on early development, however maybe it would be better to have it more briefly explained, perhaps in the form of a student drawn diagram or presented as a table. There also is a problem with the image uploaded in the early development section, so should fix that before final submission. The ‘abnormalities’ section is also done well however more conditions should be listed and described with pictures for each one. There are also only abnormalities of the kidneys listed, so maybe it would be better to have more of the other components of the renal system as well (bladder, ureter, urethra).

Also, maybe more information regarding the anatomy of the kidneys and renal system should be added, as this is an anatomy course. Some images are also missing the student image template.
Most images are uploaded correctly with the right information, maybe more would make the page look more aesthetically pleasing as well as assist learning.

Referencing is done correctly with a numbering system and in-text citations are also correct. The in-text referencing in the ‘anatomical position’ sub section of ‘fetal development’ of the ‘Kidney’ section is not referenced appropriately so just fix that minor problem.

Overall, this is great work and should just include more information in certain sections and upload more images, preferably some student drawn images as well. Well done!

====Group Project 3====

''Gastrointestinal Development''

‘GIT system overview’ section is good but requires more information to introduce the GIT and what the page is going to have information on. Timeline could form part of this section and could also preferable be in the form of a student drawn image or even a table. The overview section also contains no in-text citations. It’s a great idea to split the GIT into the three parts: foregut, midgut and hindgut to aid in understanding. There is not much information on recent findings without any mention of current models as well so perhaps it would be best to address this before final submission.

In the foregut section there is not much mention of blood supply or innervation as was done for midgut and hindgut. Student drawn images are very impressive and referenced correctly with the student template, description, title and copyright information. The features of the midgut section could include some histological drawings or images. The ‘abnormalities’ section does not contain many in-text citations in one of the paragraphs and could include more deformities listed and described with more images, as well as information on how to treat and manage such disorders later in life. There is also no information or images addressing historical findings or current models so this needs to be looked into.

The references are correctly done and ordered, and are present at the bottom of the page. Some of the in-text citations aren’t throughout the text like they should be, for example, in the stomach, liver and gallbladder, and oesophagus sections.

Overall, good effort so far but more extensive research needs to be conducted for models and findings and more information for Abnormalities, as well as a few minor edits to make the page present more nicely.

====Group Project 5====

''Integumentary Development''

This page looks very neat and well organised, with an introduction that explains exactly what is going to appear and be discussed on the page. The Development Overview section is very well done, with the appropriate use of subheadings and content. The use of dot points is very effective, making the page look neater. Perhaps it would be good to draw a histological diagram of the skin layers, and uploading it to the skin development section. Specialised cells or important names throughout the page could be highlighted in bold or underlined as well, to highlight important terms and make it easier to learn and remember from. The title ‘Some Recent Findings’ accurately portrays what we as students can only do, which is identify SOME of the recent findings. This section could have more than 2 recent findings however and could be further subdivided by subheadings into the different components of the integumentary system – perhaps have 2-3 research articles for each component of the system. Historic findings are well researched but some more information would be good. The ‘Abnormalities’ section is so far the best looking section as it seems it is almost completed. Perhaps a few more abnormalities would be even better.

The table of the timeline in the ‘Development Overview’ section is superbly done and the use of histological images is fantastic as it provides the anatomical information visually. When I clicked on an image however, there was no proper referencing of the image and the copyright information and student image information was not present. The images are described very well.
One image has a problem and is present in red writing, so might need to remove this as something is wrong with the file and it could not be uploaded. There are no student-drawn images and I think if this group did this, it would really benefit their project and emphasise their understanding.

The ‘Some Recent Findings’ section has a purple background, which makes the page look more aesthetically pleasing and less monochrome. I like the ‘More recent papers’ box that can be expanded to reveal any more research papers related to the integumentary fetal development, in case anyone wants to have a further read- very clever.

Journal articles are correctly referenced but website references need to be improved upon- to find how to do this go to the ‘How to reference’ page. References are all over the place and need to be compiled under each heading or one main heading titled ‘References’ at the bottom of the page.

Overall, this page is looking fantastic at this point in time so keep up the great work!

====Group Project 6====

''Endocrine Development''

An introduction could be very useful to summarise what the page is going to discuss. Sections 1.2-1.11 could all be subheadings under the main heading ‘System Development’, and then each of these subheading could be further divided into smaller subheadings with timeline, introduction detailing structure/ function of the endocrine organ. It is however very well done how the headings of each organ are then further subdivided into ‘abnormalities’, ‘research findings’ and ‘timeline’. However, the fact that each section has its own references and is subdivided as such, shows that even though the page may appear more ordered, there appears to be little communication between group members at this stage. So perhaps a goal could be to make the page look like one flowing work piece as opposed to sections that each person has done.

I think the content is very well researched and I like the way each organ of the endocrine system is discussed, as all are important in fetal development. The use of images is appropriate and very well done as they are referenced correctly and when you click on an image it takes you to a new page showing the student image template, copyright information as well as extra information regarding the image. There are no student-drawn images however, so perhaps it could be possible to draw a flow chart perhaps of gonadal fetal development. The use of tables is also done very well and is frequent throughout the page, with some being used to illustrate the anatomical development of certain organs, for example, the adrenal gland and pancreas. The graphs are also useful in portraying information from research findings.

The project page is missing information regarding historic findings, and I think that if this page is going to have a main heading for Abnormalities, then the group should put all their information regarding abnormalities under this section. Although it is not an endocrine organ that grows within the developing foetus, but is an important part of the mother, there is not much information on the page regarding the placenta. This section needs to be completed as the placenta is an important source of hormones and acts as an endocrine organ during the pregnancy, sustaining the foetus.

It is good that there are many references, indicating thorough research into the endocrine system with each organ heading have its own sources, however I think these references need to be ordered better. The actual referencing is done correctly, however in-text referencing is absent, so it may be best to fix this. Most images are referenced correctly as well.

Overall, keep up the good work, but just edit the page to make it look neater and finish the sections you need to.

====Group Project 7====

''Neural (CNS) Development''

This project page is very nicely organised with the group clearly specifying what aspect of neural development they are covering, being the CNS. The use of headings and subheadings is done very neatly, however sections 1.1-1.5 could be subheadings for the larger title ‘system development’. The key points have been clearly described but there is no referencing throughout the ‘Introduction’, ‘Brain development’ and ‘Abnormalities’ sections. Most key points have at least some information on them which is good for this stage of the project; however some of the headings without could use some more work.

The choice of content is highly appropriate and the use of diagrams and pictures help show the groups understanding of the project thus far. I particularly like the use of subheadings in this project as they make the page look neater and organised. The image showing the timeline of fetal neural development is good however perhaps it would be better to draw or make a timeline on the computer in order to show better understanding of the time course of fetal development. Most images that have been uploaded are also well referenced and when clicking onto them, it takes the reader to a page that has more information related to the image. The table to describe anatomical details is also done well and is important that such a key point is mentioned seeing as this is an anatomy course.

I also really like how the ‘Current research, models and findings’ section is split into ‘Current research’ and ‘Future Research’, however it seems future research needs to be further looked into. The ‘Abnormalities’ section is done very well, with multiple abnormalities listed with images used to show each one. The bolding of several key words is seen and is helpful in showing understanding of some of the key points. There are also no historic findings so try and find some information on that.

Referencing is correctly done with most references being in one main section at the end, and ordered correctly. In-cite referencing is also done correctly. All images are correctly referenced with copyright information present and the student image template. I also like the way the current research findings sources have been referenced with the use of dot points assisting learning by not just presenting to the reader as a blob of information.

Overall, well done group 7! Keep up the great work!

====Group Project 8====

''Musculoskeletal Development''

The key points of musculoskeletal development appear as headings however there is still much that needs to be clearly discussed beneath each of these points. The main headings are good and specific but some are way too specific and should be under much larger headings, for example, 1.2-1.9 could be subheadings that come under the heading ‘System Development’. ‘Background embryonic development’ is useful to understand but perhaps it is better to not have so much detail, or summarise it in a table. The ‘Abnormalities’ heading is done well, with one disease listed (Duchenne Muscular Dystrophy). It might be better to have more than one abnormality listed and clearly described as well. I particularly like the use of statistics and genetic references. It seems most of the key points relating to system development have been clearly described, but some tidying up in terms of editing needs to be done.

Also, more work needs to be done on historic findings, current research, models and findings. Once all the research parts are completed, the timeline can be correctly constructed. Also like the idea of putting a timeline and the heading shows that this is intended. More subheadings could be used to make the page look more organised and pleasing to the eye.

There are also no graphs or tables as well as pictures. A table could be used to make the timeline or highlight the differences between the second, third trimesters and neonatal periods of fetal muscular development. Maybe the initial heading of the page should be changed to ‘Muscular Fetal Development’ to indicate that muscular development is actually being covered instead of both muscular and skeletal. There also isn’t much information regarding limb fetal development, so maybe it would be good to go through that on a deeper level.

It could also help to have images loaded onto the page or to draw flow diagrams to assist in the description of how the muscles develop in the fetal period. For example, upload an image showing the difference between slow twitch and fast twitch muscle fibres or draw a flow chart to show better understanding of the molecular and cellular regulation of fetal myogenesis.

References need to be in one larger section at the end under the heading ‘References’, not two and scattered throughout as is seen. The major section of references appears to be referenced correctly and in-cite references are done very well. There are also many references which are good and show that this group has thoroughly researched their topic.

Overall, this group has done very well and just needs to add more information for certain headings, as well as organise the page a bit better in neater headings and subheadings. Pictures should be added, as well as graphs, tables and own student-drawn images.

==Online Assessment 9==

'''Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos'''.<ref name=PMID22819672><pubmed>22819672</pubmed></ref>

One of the early stages of embryonic eye development involves the invagination of the optic vesicle resulting in the formation of the bilayered optic cup with a groove on its anterior aspect. This groove is termed the optic fissure (also known as embryonic fissure) and creates an opening into which the hyaloid artery and vein can enter and exit the developing eye. As time passes, this fissure begins to fuse back together, enclosing the hyaloid vessels and this event occurs between the 6th to 7th weeks of gestation<ref name=PMID1628748><pubmed>1628748</pubmed></ref>. The zebrafish is a suitable model for observing such development due to its faster time course, so in embryos the fissure takes approximately 2 days post-fertilisation to close. When this optic fissure fails to close, a disorder known as ocular coloboma occurs, leading to impaired vision and possibly blindness later on in life<ref name=PMID16932062><pubmed>16932062</pubmed></ref>.

Further studies conducted on zebrafish and mouse models have shown genetic mutations are responsible for the abnormal patterning of the optic vesicle and decreased gene expression involving the anterior eye and periocular mesenchyme and subsequently, excess tissue cell proliferation and fusion abnormalities<ref name=PMID17609112><pubmed>17609112</pubmed></ref>. Therefore, it is evident that there are many mechanisms responsible for the fusion event of the optic fissure but no studies have previously attempted to understand the mechanisms that result in the failure of this fusion event. Hence, the present study hypothesizes that the hyaloid vasculature is somehow related to this fusion event and if there are variations in the blood vessels such as dilatations, then the optic fissure does not close properly.

The study uses zebrafish Imo2 mutants that fail to close the optic fissure at 2 days post-fertilisation. This was done by isolating RNA from Imo2 mutants (1-day post-fertilisation) and cloning it using vectors. The gene mutation was then introduced into the RNA and injected into the embryo. Once the 2-day post-fertilisation window passed, embryos were fixed and tissues were sectioned and stained. In-situ hybridisation of the embryos was performed, antibodies labeled and TUNEL performed. On another set of embryos, microangiography and imaging was performed and data quantified and analysed.

The present study searched for mutations responsible for causing ocular coloboma using a genetic screen. A mutant line designated vu270 was identified at 2 days post-fertilisation, where failure of the fusion of the optic fissure was evident. Two other phenotypes of the embryos were observed, being a larger head and no apparent red blood cells. The zebrafish injected with the mutation as stated earlier failed to generate red blood cells and thus, this study proved that the lmo2 gene has a crucial role for hematopoiesis. The study also condemns the zebrafish an appropriate model to study the roles of lmo2 in embryonic development.
In comparison to its role in red blood cell formation, the functions of lmo2 in vascular development are not as well known. Whilst previous studies demonstrated the requirement of lmo2 in angiogenesis<ref name=PMID11857074><pubmed>11857074</pubmed></ref>, the present study although reiterated this, showed abnormal formation of the blood vessels. This study further condemned the zebrafish as a relevant model to study the function of lmo2 in angiogenesis and the abnormalities associated with the vasculature that arises due to this gene, are a result of the genes’ role in maintaining vascular permeability and integrity. Therefore, it is evident that the absence of the lmo2 gene correlates with increased permeability of the vasculature.
Even more specifically, the results showed constrictions within the hyaloid artery and nerve, but severe dilatations in the hyaloid vein, indicating that lmo2 has different roles in the development of different blood vessels, or that in constricted vessels there is no flow. However, the idea that in constricted blood vessels there is no flow can be rid of as a conclusion since microangiography results showed blood flow.

In conclusion, the results of the present study demonstrate that abnormal blood vessels coursing through the optic fissure can indeed interfere with its closure.
[[Sensory - Vision Development]]

<references/>

==Online Assessment 10==

<pubmed>25085858</pubmed>

2014 Group Project 4

2014-10-24T09:17:23Z

Z3417753: /* Genital */

{{ANAT2341Project2014header}}
[[File:SexualDifferentation.jpg|350px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

=Genital=

==Introduction==

Genital system development occurs in both the embryonic and foetal phase of development. By the commencement of the foetal period, sexual determination and initial growth of the different gonads occur <ref name=Hill2014>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>. It is within the foetal period that the internal and external genital organs develop <ref name=Hill2014>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>. This page focuses on the foetal developmental processes, exploring the current and historical models and understanding, alongside the congenital abnormalities.

Today there is an innumerable amount of research articles focused on foetal genital growth, with some addressing the system as a whole <ref name=PMID14641326><pubmed>14641326</pubmed></ref> and others investigating certain genital organs <ref name=PMID13362960><pubmed>13362960</pubmed></ref>. Current models and understandings have been obtained from both human <ref name=PMID11315960><pubmed>11315960</pubmed></ref> and animal populations <ref name=PMID13362960><pubmed>13362960</pubmed></ref>, and this page will outline some of the important current research.

Research into foetal genital development can be traced to as early as the 16th century<ref name=PMID18462432><pubmed>18462432</pubmed></ref> with anatomists proposing theories and constructing models, proven to being critical in obtaining the in-depth information known today. This page will mention some of these historical findings in both the female and male genital system.

This project will end with discussing some of the congenital abnormalities of the genital system, mentioning both the malformations that are most common <ref name=PMID16006950><pubmed>16006950</pubmed></ref> and those that are rare <ref name=PMID23635766><pubmed>23635766</pubmed></ref>.

==System Development==

[[File:Paramesonephric duct.jpg|300px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|300px|thumb|Histological image of primordial follicles in infant ovary]]

'''Timeline of Embryonic, Fetal and Postnatal Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

----

===Development of Internal Genitalia===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

[[File:Kollmann454.jpg|thumb|200px|right|Internal genitalia in the female]]

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
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[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
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===Descent of the Gonads===

[[File:Testis-descent end.jpg|200px|thumb|right|Descent of testes]]

'''Male:''' The testis undergo transabdominal and transinguinal descent. The testis lies in the subserous fascia. The processus vaginalis evaginates into the scrotum and the gubernaculum draws it into the scrotal sac. As it descends, it traverses past the superficial (external oblique fascia) and deep (transversalis fascia) inguinal rings of the inguinal canal. This begins in between the 4th and 5th week (usually around day 26) and spans over many days. It can occur unilaterally or bilaterally, more common in premature babies and can conclude postnatally.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

'''Female''': ovaries undergo caudal and lateral shifts to be suspended in the broad ligament of the uterus. The gubernaculum does not shorten, attaches to paramesonephric ducts and causes medial translocation into the pelvis. The remnant in adult life of the gubernaculum is the ovarian and round ligament of the uterus, which suspend the ovaries and uterus respectively.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

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==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
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{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

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{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
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'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
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'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

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{|
|-bgcolor="lavenderblush"
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'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
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'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
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{|
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'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

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{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T09:11:35Z

Z3417753: /* Genital */

2014 Group Project 4

2014-10-24T08:57:42Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Genital system development occurs in both the embryonic and foetal phase of development. By the commencement of the foetal period, sexual determination and initial growth of the different gonads occur <ref name=Hill2014>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>. It is within the foetal period that the internal and external genital organs develop <ref name=Hill2014>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>. This page focuses on the foetal developmental processes, exploring the current and historical models and understanding, alongside the congenital abnormalities.

Today there is an innumerable amount of research articles focused on foetal genital growth, with some addressing the system as a whole <ref name=PMID14641326><pubmed>14641326</pubmed></ref> and others investigating certain genital organs <ref name=PMID13362960><pubmed>13362960</pubmed></ref>. Current models and understandings have been obtained from both human <ref name=PMID11315960><pubmed>11315960</pubmed></ref> and animal populations <ref name=PMID13362960><pubmed>13362960</pubmed></ref>, and this page will outline some of the important current research.

Research into foetal genital development can be traced to as early as the 16th century<ref name=PMID18462432><pubmed>18462432</pubmed></ref> with anatomists proposing theories and constructing models, proven to being critical in obtaining the in-depth information known today. This page will mention some of these historical findings in both the female and male genital system.

This project will end with discussing some of the congenital abnormalities of the genital system, mentioning both the malformations that are most common <ref name=PMID16006950><pubmed>16006950</pubmed></ref> and those that are rare <ref name=PMID23635766><pubmed>23635766</pubmed></ref>.

==System Development==

[[File:Paramesonephric duct.jpg|300px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|300px|thumb|Histological image of primordial follicles in infant ovary]]

'''Timeline of Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

----

===Development of Internal Genitalia===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

[[File:Kollmann454.jpg|thumb|200px|right|Internal genitalia in the female]]

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[[File:External genitalia current model.jpg|400px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

===Descent of the Gonads===

[[File:Testis-descent end.jpg|200px|thumb|right|Descent of testes]]

'''Male:''' The testis undergo transabdominal and transinguinal descent. The testis lies in the subserous fascia. The processus vaginalis evaginates into the scrotum and the gubernaculum draws it into the scrotal sac. As it descends, it traverses past the superficial (external oblique fascia) and deep (transversalis fascia) inguinal rings of the inguinal canal. This begins in between the 4th and 5th week (usually around day 26) and spans over many days. It can occur unilaterally or bilaterally, more common in premature babies and can conclude postnatally.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

'''Female''': ovaries undergo caudal and lateral shifts to be suspended in the broad ligament of the uterus. The gubernaculum does not shorten, attaches to paramesonephric ducts and causes medial translocation into the pelvis. The remnant in adult life of the gubernaculum is the ovarian and round ligament of the uterus, which suspend the ovaries and uterus respectively.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T08:53:04Z

Z3417753: /* Development of Internal Genitalia */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Genital system development occurs in both the embryonic and foetal phase of development. By the commencement of the foetal period, sexual determination and initial growth of the different gonads occur <ref name=Hill2014>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>. It is within the foetal period that the internal and external genital organs develop <ref name=Hill2014>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>. This page focuses on the foetal developmental processes, exploring the current and historical models and understanding, alongside the congenital abnormalities.

Today there is an innumerable amount of research articles focused on foetal genital growth, with some addressing the system as a whole <ref name=PMID14641326><pubmed>14641326</pubmed></ref> and others investigating certain genital organs <ref name=PMID13362960><pubmed>13362960</pubmed></ref>. Current models and understandings have been obtained from both human <ref name=PMID11315960><pubmed>11315960</pubmed></ref> and animal populations <ref name=PMID13362960><pubmed>13362960</pubmed></ref>, and this page will outline some of the important current research.

Research into foetal genital development can be traced to as early as the 16th century<ref name=PMID18462432><pubmed>18462432</pubmed></ref> with anatomists proposing theories and constructing models, proven to being critical in obtaining the in-depth information known today. This page will mention some of these historical findings in both the female and male genital system.

This project will end with discussing some of the congenital abnormalities of the genital system, mentioning both the malformations that are most common <ref name=PMID16006950><pubmed>16006950</pubmed></ref> and those that are rare <ref name=PMID23635766><pubmed>23635766</pubmed></ref>.

==System Development==

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]]

'''Timeline of Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

----
===Development of Internal Genitalia===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

[[File:Kollmann454.jpg|thumb|200px|right|Internal genitalia in the female]]

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[[File:External genitalia current model.jpg|400px|centre|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

===Descent of the Gonads===

[[File:Testis-descent end.jpg|200px|thumb|right|Descent of testes]]

'''Male:''' The testis undergo transabdominal and transinguinal descent. The testis lies in the subserous fascia. The processus vaginalis evaginates into the scrotum and the gubernaculum draws it into the scrotal sac. As it descends, it traverses past the superficial (external oblique fascia) and deep (transversalis fascia) inguinal rings of the inguinal canal. This begins in between the 4th and 5th week (usually around day 26) and spans over many days. It can occur unilaterally or bilaterally, more common in premature babies and can conclude postnatally.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

'''Female''': ovaries undergo caudal and lateral shifts to be suspended in the broad ligament of the uterus. The gubernaculum does not shorten, attaches to paramesonephric ducts and causes medial translocation into the pelvis. The remnant in adult life of the gubernaculum is the ovarian and round ligament of the uterus, which suspend the ovaries and uterus respectively.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T06:07:02Z

Z3417753: /* Genital */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]]

'''Timeline of Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Development of Internal Genitalia===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

===Descent of the Gonads===

[[File:Testis-descent end.jpg|200px|thumb|right|Descent of testes]]

'''Male:''' The testis undergo transabdominal and transinguinal descent. The testis lies in the subserous fascia. The processus vaginalis evaginates into the scrotum and the gubernaculum draws it into the scrotal sac. As it descends, it traverses past the superficial (external oblique fascia) and deep (transversalis fascia) inguinal rings of the inguinal canal. This begins in between the 4th and 5th week (usually around day 26) and spans over many days. It can occur unilaterally or bilaterally, more common in premature babies and can conclude postnatally.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

'''Female''': ovaries undergo caudal and lateral shifts to be suspended in the broad ligament of the uterus. The gubernaculum does not shorten, attaches to paramesonephric ducts and causes medial translocation into the pelvis. The remnant in adult life of the gubernaculum is the ovarian and round ligament of the uterus, which suspend the ovaries and uterus respectively.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T06:02:33Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]]

'''Timeline of Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Development of Internal Genitalia===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

===Descent of the Gonads===

[[File:Testis-descent end.jpg|200px|thumb|right|Descent of testes]]

'''Male:''' The testis undergo transabdominal and transinguinal descent. The testis lies in the subserous fascia. The processus vaginalis evaginates into the scrotum and the gubernaculum draws it into the scrotal sac. As it descends, it traverses past the superficial (external oblique fascia) and deep (transversalis fascia) inguinal rings of the inguinal canal. This begins in between the 4th and 5th week (usually around day 26) and spans over many days. It can occur unilaterally or bilaterally, more common in premature babies and can conclude postnatally.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

'''Female''': ovaries undergo caudal and lateral shifts to be suspended in the broad ligament of the uterus. The gubernaculum does not shorten, attaches to paramesonephric ducts and causes medial translocation into the pelvis. The remnant in adult life of the gubernaculum is the ovarian and round ligament of the uterus, which suspend the ovaries and uterus respectively.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

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{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T06:00:00Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]]

'''Timeline of Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Development of Internal Genitalia===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

===Descent of the Gonads===

[[File:Testis-descent end.jpg|200px|thumb|right|Descent of testes]]

'''Male:''' The testis undergo transabdominal and transinguinal descent. The testis lies in the subserous fascia. The processus vaginalis evaginates into the scrotum and the gubernaculum draws it into the scrotal sac. As it descends, it traverses past the superficial (external oblique fascia) and deep (transversalis fascia) inguinal rings of the inguinal canal. This begins in between the 4th and 5th week (usually around day 26) and spans over many days. It can occur unilaterally or bilaterally, more common in premature babies and can conclude postnatally.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>

'''Female''': ovaries undergo caudal and lateral shifts to be suspended in the broad ligament of the uterus. The gubernaculum does not shorten, attaches to paramesonephric ducts and causes medial translocation into the pelvis. The remnant in adult life of the gubernaculum is the ovarian and round ligament of the uterus, which suspend the ovaries and uterus respectively.<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T05:42:00Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]]
[[File:Testis-descent end.jpg|200px|thumb|Descent of testes]]

'''Timeline of Genital Development'''<ref>Hill, M.A. (2014) Lecture - Genital Development. Retrieved October 24, 2014, from https://embryology.med.unsw.edu.au/embryology/index.php/Lecture_-_Genital_Development</ref>
{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T05:25:49Z

Z3417753: /* Genital */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]]
[[File:Testis-descent end.jpg|200px|thumb|Descent of testes]]

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T04:55:26Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

[[File:Paramesonephric duct.jpg|300px|thumb|Paramesonephric duct development]]
[[File:Infant ovary.jpg|300px|thumb|Histological image of primordial follicles in infant ovary]]
[[File:Testis-descent end.jpg|300px|thumb|Descent of testes]]

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
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| style="width:50%"|
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[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

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==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
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'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
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'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

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{|
|-bgcolor="lavenderblush"
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'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
|-bgcolor="lavenderblush"
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'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

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{|
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'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T04:45:28Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins
|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

[[File:Paramesonephric duct.jpg|200px|thumb|Paramesonephric duct development]][[File:Infant ovary.jpg|200px|thumb|Histological image of primordial follicles in infant ovary]][[File:Testis-descent end.jpg|200px|thumb|Descent of testes]]

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T04:38:27Z

Z3417753: /* Development of the External Genitalia */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Findings==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for Historical Findings

==Abnormalities==

Click [https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9 here] for foetal genital abnormalities

==References==
<references/>

2014 Group Project 4

2014-10-24T02:16:03Z

Z3417753: /* Genital */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

'''Related video'''

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T02:08:04Z

Z3417753: /* Male */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf | Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T02:05:44Z

Z3417753: /* Current Models */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
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[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
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===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf | Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
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{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

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{|
|-bgcolor="aliceblue"
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[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
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'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
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'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
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====Female====
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{|
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'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
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{|
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'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
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{|
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'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
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'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T01:21:51Z

Z3417753: /* Female */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf| Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

[[File:Human Y chromosome SRY region.jpg|400px|right|thumb| Human Y chromosome showing SRY gene]]

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T01:18:25Z

Z3417753: /* Male */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf| Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

[[File:Male testosterone and AMH level graph.jpg|thumb|300px|right|Graph showing male testosterone and AMH levels]]

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T01:16:37Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple"|align="centre"
|'''Week''' || '''Development'''|||'''Image'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|[[File:Paramesonephric duct.jpg|200px]]
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|[[File:Infant ovary.jpg|200px]]
[[File:Testis-descent end.jpg|200px]]
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf| Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

File:SexualDifferentation.jpg

2014-10-24T01:01:04Z

Z3417753: /* =Copyright */

==Sexual Differentiation==
The stages in sexual differentiation of the female and male reproductive system

==Reference==
http://en.wikipedia.org/wiki/Sexual_differentiation
==Copyright==
©CC BY 3.0
http://creativecommons.org/licenses/by/3.0/

{{Template:Student Image}}

2014 Group Project 4

2014-10-24T00:59:28Z

Z3417753: /* System Development */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{| class="wikitable"
|-bgcolor="purple" align="centre"
|'''Week''' || '''Development'''
|-bgcolor="lavender"
| 3-4 || Primordial germ cells migrate during gastrulation
|-
| 4 || Intermediate mesoderm, pronephros primordium
|-bgcolor="lavender"
| 5 || Mesonephros and mesonephric duct
|-
| 6 || Ureteric bud, metanephros, genital ridge
|-bgcolor="lavender"
| 7 || Cloacal divison, gonadal primordium - indifferent to first appearance of testis cords
Female - Paramesonephric duct preservation or regression begins

|-
| 8 || Paramesonephric duct, clear gonadal differentiation
|-bgcolor="lavender"
| 9 || Paramesonephric duct fusion in the female forming the uterus and lack of fusion laterally forming the fallopian tubes. Also forms vagina.
|-
| 15 || Primary follicles (ovary)
From the 26th week (between week 4 and 5), the gubernaculum starts to pull the testes down and results in descent of the testes into the scrotal sac.
Both male and female gonads undergo descent.
|-bgcolor="lavender"
| Puberty || Development of secondary sexual characteristics
Female - start of menstruation, first egg released.
Male - development of course voice, body hair and sperm formation.
|-
|}

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

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===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf| Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

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{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

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{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

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==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T00:27:38Z

Z3417753: /* Current Models */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
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[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

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===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

All animal experiments must be performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. [[http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf| Guide for the Care and Use of Laboratory Animals]]

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

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{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

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{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

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{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

File:POPs and risk of hypospadias.jpg

2014-10-24T00:21:59Z

Z3417753:

===Table of Maternal Serum Concentrations of PCB-153, p,p'-DDE and HCB during early pregnancy (between12th and 14th weeks of gestation) and risk of hypospadias in infants===

This table shows that the EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias.

====References====
<pubmed>23028613</pubmed>

====Copyright====
© Rignell-Hydbom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Template:Student Image}}

2014 Group Project 4

2014-10-24T00:18:17Z

Z3417753: /* Male */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

[http://npesu.unsw.edu.au/sites/default/files/npesu/surveillances/Congenital%20anomalies%20in%20Australia%202002-2003.pdf| Statistics regarding congenital abnormalities, including hypospadias and epispadias for 2002 and 2003 in Australia]

----

{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

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{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T00:11:34Z

Z3417753: /* Female */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

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===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-24T00:08:49Z

Z3417753: /* Male */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

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===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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{|
|-bgcolor="aliceblue"
|
[[File:POPs and risk of hypospadias.jpg|400px|thumb|left|Table of levels of POPs in maternal serum samples and risk of hypospadias in infants]]

'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

File:POPs and risk of hypospadias.jpg

2014-10-24T00:07:09Z

Z3417753:

===Table of Maternal Serum Concentrations of PCB-153, p,p'-DDE and HCB during early pregnancy (between12th and 14th weeks of gestation) and risk of hypospadias in infants===

====References====
<pubmed>23028613</pubmed>

====Copyright====
© Rignell-Hydbom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Template:Student Image}}

File:POPs and risk of hypospadias.jpg

2014-10-24T00:05:57Z

Z3417753: ===Table of Maternal Serum Concentrations of PCB-153, p,p'-DDE and HCB during early pregnancy=== ====References==== <pubmed>23028613</pubmed> ====Copyright==== © Rignell-Hydbom et al. This is an open-access article distributed under the terms of the...

===Table of Maternal Serum Concentrations of PCB-153, p,p'-DDE and HCB during early pregnancy===

====References====
<pubmed>23028613</pubmed>

====Copyright====
© Rignell-Hydbom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Template:Student Image}}

2014 Group Project 4

2014-10-23T23:59:09Z

Z3417753: /* Male */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
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{|
|-bgcolor="aliceblue"
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'''A Nested Case-Control Study of Intrauterine Exposure to Persistent Organochlorine Pollutants and the Risk of Hypospadias (2012)'''<ref name=PMID23028613><pubmed>23028613</pubmed></ref>

Hypospadias are a common congenital abnormality resulting from the failure of fusion of the urethral folds within the fetal period of development (8th-14th week of gestation). Whilst it is known that sexual differentiation of the male external genitalia depends on testosterone and its conversion into dihydrotestosterone, the risk factors of hypospadias are not thoroughly researched. This article aims to examine environmental exposure to endocrine disrupting chemicals (EDCs) and understand their potential to act as antagonists on androgen receptors, thereby disrupting the hormonal balance of the endocrine system ultimately leading to hypospadias.<ref name=PMID11469497><pubmed>11469497</pubmed></ref>
More specifically, a major group of EDCs known as persistent organochlorine pollutants (POPs), for example, polychlorinated biphenyls, dioxins, pesticides (dichlorodiphenyl trichloroethane, DDT) and hexachlorobenzene (HCB). Such chemicals are lipophilic, resistant to biodegradation and are present throughout the hydrosphere and atmosphere. Although these chemicals were banned in the 1970’s and 1980’s, due to the potency of these chemicals, they are still found within humans<ref name=PMID22425898><pubmed>22425898</pubmed></ref> and are able to traverse the placenta, becoming exposed to the developing fetus<ref name=PMID6431068><pubmed>6431068</pubmed></ref>.
Therefore, the aim of the present study was to investigate the linkage between exposures of the fetus to POPs and risk of developing hypospadias.

This was a case-controlled study of the risk of hypospadias of single-born boys with regards to levels of POPs within the mothers’ blood during pregnancy. The study used 390 boys with hypospadias and controls were used. Boys with any cryptorchidisms, major malformations or even minor hypospadias were excluded from the study.

This study concluded that EDCs such as PCBs, p,p’-DDE and HCB possess the potential to alter hormonal levels and affect the fetus, resulting hypospadias. It shows that in uterine exposure to HCB and possibly p,p’-DDE are risk factors and may affect androgen-signaling.
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{|
|-bgcolor="aliceblue"
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'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
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{|
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'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
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====Female====
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{|
|-bgcolor="lavenderblush"
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'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
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{|
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'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
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{|
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'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
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{|
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'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
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{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

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==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T22:55:23Z

Z3417753:

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system.

==System Development==

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – Genital Development''
|- bgcolor="lavender"
|
# Genital System development begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally on the coelomic cavity
# The proliferation of this coelomic epithelium leads to an outgrowth due to the ingression of the coelomic epithelium, proliferation and recruitment of adjacent mesonephric cells
# This bipotential genital ridge is indifferentiated in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively
|}

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression. <ref name=PMID11315960><pubmed>11315960</pubmed></ref> <ref name=PMID24240231><pubmed>24240231</pubmed></ref>

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the '''epididymis''' <ref name=PMID24240231><pubmed>24240231</pubmed></ref>. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate. <ref name=PMID24240231><pubmed>24240231</pubmed></ref>
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings. <ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID11315960><pubmed>11315960</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
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===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

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{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
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{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

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{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

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{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

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{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

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==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T13:46:44Z

Z3417753:

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system..

==System Development==

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
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[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

{|
|-bgcolor="lavenderblush"
|
'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.
|}

----

{|
|-bgcolor="lavenderblush"
|
'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.
|}

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T13:37:14Z

Z3417753: /* Current Research and Findings */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system..

==System Development==

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

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{|
|-bgcolor="aliceblue"
|
'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|}

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|}

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

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==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T13:32:37Z

Z3417753: /* Male */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system..

==System Development==

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----
{|
|-bgcolor="aliceblue"
|
[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.
|]

----

{|
|-bgcolor="aliceblue"
|
'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.
|]

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T13:08:06Z

Z3417753: /* Introduction */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system..

==System Development==

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T13:05:32Z

Z3417753: /* Genital */

{{ANAT2341Project2014header}}
=Genital=
==Introduction==

[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

This page will explain the development of the genital system in the fetal stage of development, of both males and females, after briefly explaining the embryonic development. It explores the models of fetal development of the internal and external genitalia, including the timeline of development. This page also lists some animal models used in research and outlines some current research and findings regarding the development of the genital system, including historic findings. Finally, this page also discusses some of the congenital abnormalities of the genital system..

==System Development==

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

===Development of the External Genitalia ===

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T12:38:16Z

Z3417753: /* Female */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name= Abbott>Abbott David, H. '''Fibrillin-3 in the fetal ovary: can it contribute to polycystic ovary syndrome?'''. Expert Review of Endocrinology & Metabolism: 2012, 7(1); 31-34</ref>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T12:19:24Z

Z3417753: /* Current Research and Findings */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[File:Ultrasound male.jpg|200px|thumb|left|Ultrasound of male fetus]]

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name=PMID23585338><pubmed>23585338</pubmed></ref>

<pubmed>23585338</pubmed>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

File:Ultrasound male.jpg

2014-10-23T12:12:17Z

Z3417753:

===Ultrasound determining gender - boy===

Image shows an ultrasound of fetus with a penis and testes, indicating the male gender of the baby.

====Reference====

Image URL: http://upload.wikimedia.org/wikipedia/commons/a/a1/Boy.JPG

Page URL:http://en.wikipedia.org/wiki/Obstetric_ultrasonography#Fetal_sex_discernment

Author: X.Compagnion

====Copyright====
Permission is granted to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later version published by the Free Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts. A copy of the license is included in the section entitled GNU Free Documentation License.

{{Template:Student Image}}

File:Ultrasound male.jpg

2014-10-23T12:11:08Z

Z3417753: ===Ultrasound determining gender - boy=== Image shows an ultrasound of fetus with a penis and testes, indicating the male gender of the baby. ====Reference==== Image URL: http://upload.wikimedia.org/wikipedia/commons/a/a1/Boy.JPG Page URL:http://en...

2014 Group Project 4

2014-10-23T12:08:40Z

Z3417753: /* Female */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

[[F

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name=PMID23585338><pubmed>23585338</pubmed></ref>

<pubmed>23585338</pubmed>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T11:32:48Z

Z3417753: /* Development of the External Genitalia */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercle itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name=PMID23585338><pubmed>23585338</pubmed></ref>

<pubmed>23585338</pubmed>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T11:28:18Z

Z3417753: /* Development of the External Genitalia */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* The tubercles itself goes on to form the mons pubis.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name=PMID23585338><pubmed>23585338</pubmed></ref>

<pubmed>23585338</pubmed>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T11:22:12Z

Z3417753: /* Development of the External Genitalia */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
|- style="height:10px"
| style="width:50%"|
|}

===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|600px|thumb|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name=PMID23585338><pubmed>23585338</pubmed></ref>

<pubmed>23585338</pubmed>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

2014 Group Project 4

2014-10-23T11:18:55Z

Z3417753: /* Development of the External Genitalia */

{{ANAT2341Project2014header}}
=Genital=
==System Development==

[[File:SexualDifferentation.jpg|300px|right|thumb|The stages in sexual differentiation of the female and male reproductive system]]

Prior to discussing the fetal development of the genital system, it is important to briefly appreciate the embryonic development of the genital tracts. Up until the seventh week of development, male and female genital tracts are '''indifferent'''. The genital system begins from a thickening of the epithelium surrounding the mesonephros, which lies dorsally in the coelomic cavity. The proliferation of this coelomic epithelium leads to an outgrowth known as the '''genital ridge'''. The genital ridge continues in outgrowth due to the ingression of the polemic epithelium, proliferation and recruitment of adjacent mesonephric cells. This bipotential genital ridge is indifferent in XX and XY embryos, however due to chromosomal and genetic influences differentiate into the testis and ovary respectively. [1]

Fetal genital development occurs in three main stages
1. Differentiation of gonad into Ovary or Testis

===Internal Genital Development===
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE'''
|bgcolor="violet"|'''FEMALE'''
|-
|bgcolor="aliceblue"| The fetal development of internal genitalia is largely dependent on the endocrine functions of the fetal testes. The fetal testes produce masculinizing hormones such as '''testosterone''' which begins its release from the interstitial Leydig cells of the primitive seminiferous tubules during the 8th week of development, and also the release of '''Mullerian Inhibiting Hormone (MIS)''' which is released at the sixth and seventh weeks by the Sertoli Cells. Testosterone acts primarily on the mesonephric ducts to stimulate the formation of the male genital ducts, whereas the MIS acts on the paramesonephric duct to stimulate its regression.

By the eighth week of fetal development in XY embryos, the testosterone produced in the testes results in the convolution of the proximal ends of the mesonephric ducts to form the 'epididymis. The mesonephros begins to degenerate, however some of the mesonephric tubules remain and develop into efferent ductules, which then open into the duct of the epididymis. Distal to this end, the mesonephric duct begins to develop a thick lining of smooth muscle and progresses to become the ductus deferens.

''Fetal Male Glandular Development''
* the seminal glands develop from lateral outgrowths from that caudal end of each mesonephric duct which nourishes the sperm and constitutes most of the fluid in the ejaculate
* the endodermal outgrowths arise from the prostatic part of the urethra which grow into the surrounding mesenchyme. This acts as a base of the proliferation of the glandular epithelium of the prostate to differentiate- the associated mesenchyme will differentiate into dense stroma and the smooth muscle of the prostate.
* The bulbourethral glands are pea-sized and developed from paired outgrowths that originate from the spongy pat of the urethra. The adjacent mesenchyme gives rise to the stroma and smooth muscle fibres which will ultimately produce secretions which contribute to the semen.

| bgcolor="lavenderblush"| The absence of testosterone production in XX embryos results in the regression of the mesonephric duct, and conversely the absence of MIH results in the development and progression of the paramesonephric ducts. The paramesonephric ducts give rise to most of the female internal genital system- the unfused cranial aspects of the ducts give rise to a primitive '''fallopian tubes''', whilst the caudally fused portions form the uterovaginal primordium which will develop into a '''uterus''' and '''superior vagina'''. The splanchnic mesenchyme gives rise to the endometrial stromal tissue and the myometrium.

Female genital development during the fetal period is not dependant on endocrine contributions from the foetus. Later in fetal development, maternal oestrogen's and oestrogen derived from the placenta contribute to the development of the fallopian tubes, uterus and the superior vagina.

''Female Glandular Development''

* The urethra develops outgrowths which form the mucus secreting '''urethral glands''' and paraurtehral glands.
* outgrowths from the urogenital sinus form the '''greater vestibular glands''' in the lower third of the Labia Majora. These glands are also mucous secreting and are comparative to the bulbourethral glands in males.
|-
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===Development of the External Genitalia ===

[[File:External genitalia current model.jpg|300px|right|thumb|Flow Diagram of the current model of embryonic and fetal development of the external genitalia]]

'''The current model for embryonic development of the external genitalia in humans and mice:'''

{|class="wikitable mw-collapsible mw-collapsed"
! ''Embryonic Period – fertilisation to end of 8th week (embryonic age) = AMBISEXUAL STAGE''
|- bgcolor="lavender"
|
# The external genitalia initially begin in the perineal region as three primordia, being the genital tubercle in the midline and the bilateral genital swellings. These three primordia arise together with the differentiation of the cloacal part of the hindgut into the urogenital sinus, rectum and anal canal. The cloacal membrane extends from the perineum cranially to the root of the umbilical cord and during development, this bilayered cloacal membrane retracts into the perineum. This is due to cranial and medial migration of mesodermal cells into the ventral body wall between the ectoderm and endoderm of the cloacal membrane. These migrating mesodermal cells line around the membrane and accumulate, forming the three primordial swellings.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# These external features are internally related to the cloaca, which becomes divided coronally by the urorectal septum into the urogenital sinus anteriorly, and the rectum and anus posteriorly.
# This division of the cloaca occurs in a specific way so the allantois, Mullerian and Wolffian ducts and ureters all empty into the urogenital sinus.
# When the cloacal membrane becomes divided into the urogenital and anal membranes, the urogenital membrane is bound cranially by the genital tubercle in the midline and laterally by the urogenital folds and genital swellings.
# The urogenital membrane degenerates to allow communication between the urogenital sinus and amniotic cavity.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
|}

----

'''The current model for fetal development of the external genitalia in humans and mice:'''
{| style="width:100%"
|bgcolor="lightskyblue"|'''MALE MODEL'''
|bgcolor="violet"|'''FEMALE MODEL'''

|-
|bgcolor="aliceblue"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Initially, the female and male fetuses’ external genitalia are identical and include the genital tubercle in the midline, urogenital folds (forming the urogenital ostium) and genital swellings (laterally).
# In males, the genital tubercle will eventually form the penis and the genital swellings migrate caudally and a fusion event in the midline occurs, thus forming the scrotum.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# As the genital tubercle elongates to form the penis, a groove forms on the ventral surface known as the urethral groove. The urethral folds that are continuous with the urogenital folds surrounding the urogenital ostium define the urethral groove laterally.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
# At first, the urethral groove and folds extend only part of the along the shaft of the elongating genital tubercle (known as the phallus at this stage).
# Distally, the urethral groove terminates at the urethral plate, consisting of epithelial cells, and then extends into the glans of the penis, forming a channel.
# As the phallus elongates, the urethral folds grow toward each other and fuse in the midline forming the midline epithelial seam, converting the urethral groove into a tubular penile urethra. The fusion of the urethral folds begins proximally in the perineal region and extends distally towards the glans of the penis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Cross section of genital tubercle male.jpg|270px|right|thumb|Cross section of the male genital tubercle]][[File:Anatomical diagram of testes.jpg|300px|left|thumb|Anatomical drawing of adult male testes]]

* Hypospadias result from failure of formation or fusion of the urethral folds and this is the focus of current research.
* The elongating phallus is covered externally by ectoderm that will eventually give rise to the penile epidermis.
* Urethral epithelium has endodermal origins and the majority of the penis is derived from mesodermal cells.
* During development, the mesoderm separates into connective tissues and dermis.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dense areas of mesenchymal cells form within the shaft of the penis with the most superficial dense bodies forming the thick connective tissue capsule known as the tunica albuginae.

* Mesenchyme surrounding the urethra forms smooth muscle of the urethral mucosa and submucosa. Erectile tissues such as the corpus spongiosum and corpus cavernosum then surround these two layers.
* In some species, the mesenchyme of the genital tubercle also forms an os penis, comprised of bone and cartilage.
* Genital tubercle development involves an outgrowth of somatic tissue from the body surface, similar to the development of the limb.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Development of the external genitalia is highly regulated by the endocrine system. Sexual differentiation of the external genitalia is determined by the presence or absence of androgen receptor signaling. The fetal testes produce testosterone, which travels to the genital tubercle via the bloodstream, where it is converted into 5a-dihydrotestosterone by the enzyme 5a-reductase. This formation of the highly potent 5a-dihydrotestosterone masculinizes the developing external genitalia, as binding of the 5a-dihydrotestosterone to its androgen receptor leads to the regulation of downstream signaling genes.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
*: Sonic Hedgehog (SHH) acts as an endodermal signal that normally regulates patterning of the hindgut and is expressed in the epithelium of the cloaca, urogenital sinus and urethral plate epithelium. However this has an important signaling pathway role in development of external genitalia. The SHH gene codes for a particular protein that has important roles in organogenesis as well as structures that are dependent upon mesenchymal-epithelial interactions, such as limbs, teeth and prostate.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
| bgcolor="lavenderblush"|'''''Fetal Period – from 8th week of development = SEXUAL DIFFERENTIATION'''''
# Only minor changes occur from the embryonic ambisexual stage in the female, beginning with the minimal growth of the genital tubercle to form the clitoris.
# The urogenital folds remain apart and unfused to form the labia majora.
# The genital swellings also remain apart and unfused to form the labia minora.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

* Together, these bilateral labial structures and the clitoris located proximally form the border for the urogenital ostium, thus forming the vestibule of the vagina, with vaginal and urethral openings.
* Sexual dimorphism of the external genitalia in female humans is determined by the absence of androgenic pathways, however the female genital tubercle can be ‘masculinised’ as 5a-reductase and androgen recpetors are present.
* The genital tubercle can not go on to form a penis as testosterone is not produced, however in some instances it is abnormally produced in excess by the suprarenal gland resulting in different degrees of masculinsation of the clitoris.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

[[File:Clitoris.jpg|400px|center|Stages in the Development of the External Sexual Organs in the Male and Female]]
|-
|- style="height:10px"
| style="width:50%"|
|}

----

Until the seventh week of development, the external genitalia of both XX and XY embryos is indistinguishable,. Sexual characteristics only become apparent after the ninth week, however are only clearly different between male and female after the twelfth week of development. In the fourth week of the embryonic phase of development, the cranial end of the cloacal membrane begins to swell and proliferate and produce a protrusion called the ‘”genital tubercle”’. This protrusion acts as a precursor for the major sexual organs, the glans penis in males and the glands clitoris in females.

Also in the forth week, swellings begin to form around the cloacal membrane called the labioscrotal swellings and urogenital folds. The genital tubercle elongates in both sexes and forms a primordial phallus. The urogenital membrane which lies on the floor of the cleft formed by the urethral folds

“’Male”’
Testosterone produced by the interstitial Leydig cells of the fetal testes stimulates the masculinization of this indifferent genitalia. It stimulates the phallus to enlarges and elongates to form the “’glans penis”’, and the urethral folds begin to form the lateral walls of the urethral groove on the ventral aspect of the penis. A proliferation of endodermal cells lines the urethral groove and is referred to as the urethral plate, and it extends from the phallic portion of the urogenital sinus.

The urethral fold fuse with each other to form the ‘”spongy urethra”’ on the ventral surface of the penis. As the surface endoderm fuses in the median plane of the penis it encloses the spongy urethra- this fusion is called the “’penile raphe”’.
At the tip of the penis, the an endodermal ingrowth forms a cellular endodermal chord which grows inside the penis towards the base. This cord canalizes and the lumen joins the previously formed spongy urethra, thus completing the terminal part of the urethra, mobbing the external urethral orifice to the tip of the penis.

During the twelfth week of development, an ectodermal circular ingrowth develops around the periphery of the penis and sits like a cap. The ingrowth breaks down and forms the ‘”prepuce”’ (foreskin) which remains as a fold of skin covering the tip of a penis. The mesenchyme of the phallus gives rise to the corpus cavernosum and the corpus spongiosum. The labioscrotal swellings grow toward eachother and fuse in the center to form the “’scrotum”’, this line of fusion is eferred to as the “’scrotal raphe”’.

“’Female”’
Much like the male genitalia, the genital tubercle swells and elongates to form the “’glans clitoris”’. At eighteen weeks, the clitoris is still a prominent swelling. In females, the urethral folds only fuse at the posterior aspect and form the ‘”labia minora’’. The labioscrotal folds fuse only at two points, posteriorly to form the posterior labial commissure, and anteriorly to form the “’mons pubis”’, the remain unfused portions remain as the “’labia majora”’.

'''References'''

[1] <pubmed>PMC3841730</pubmed>
<pubmed>11315960</pubmed>

<pubmed>24240231</pubmed>
<pubmed>24928207</pubmed>
<pubmed>24741072</pubmed>

===Related video===

<html5media>https://www.youtube.com/watch?v=MureNA-RSZM</html5media>

==Current Research, Models and Findings==

===Current Models===

Most current research uses mouse models and observes the development of their external genitalia, especially their penile development, which initially appears to be different to human development. However, more microscopic inspection shows that mice have very similar external genitalia and are therefore appropriate animal models for observing such fetal development. As a result, mutant mouse models can effectively be used in future research to observe molecular mechanisms underlying hypospadias and their aetiology. <ref name=PMID14641326><pubmed>14641326</pubmed></ref>

When observing the morphology and cell biology of the developing testis, it is important to note that most of the research conducted on the subject involves the use of mouse models as a result of a lack of human subjects. It can be assumed that events in the human embryo correspond to the same events in the mouse embryo, however there are some differences between the time course of certain events and anatomy. <ref name=PMID17237341><pubmed>17237341</pubmed></ref>

Some examples of different animal models used in research involving fetal development of male and female genital systems are:

* Sheep<ref name=PMID21223560><pubmed>21223560</pubmed></ref>
* Mouse<ref name=PMID14641326><pubmed>14641326</pubmed></ref>
* Dogs<ref name=PMID13362960><pubmed>13362960</pubmed></ref>
* Porcine<ref name=PMID23571006><pubmed>23571006</pubmed></ref>
* Rats<ref name=PMID22248293><pubmed>22248293</pubmed></ref>
* Monkeys<ref name=PMID21710394><pubmed>21710394</pubmed></ref>
* Bovine<ref name=PMID20347535><pubmed>20347535</pubmed></ref>
* Goats<ref name=PMID22006251><pubmed>22006251</pubmed></ref>

Different models are used for different research topics; for example, research involving Polycystic ovary syndrome (PCOS) uses murine models preferable due to the developmental time frame that allows studies of inherited PCOS to be examined within an appropriate time frame. The sheep model is also beneficial to use for PCOS research as it is cost effective and their size allows them to be subjected to certain procedures such as ultrasound and neurotransmitter measures. Primates are examples of an optimal model however are limited in their accessibility and long time frame of development.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

It is important to note that any findings associated with an animal model should be translated appropriately to the human model, as animals differ in their anatomy and regulatory mechanisms, as well as placentation. That is, sheep, rats and mice display different placentation and ovarian development that occurs in utero in sheep, primates and humans differs to the ex utero development in murine models.<ref name=PMID21710394><pubmed>21710394</pubmed></ref>

===Current Research and Findings===
====Male====
----

[[File:Hypospadias.jpg|400px|thumb|right|Different types of hypospadias]]

Extensive research into organogenesis of the external genitalia, mainly in males, is driven by the increasing incidence of hypospadias. Hypospadias are a result of the defect of fusion of the urethral folds of the lower part of the penis to fold and form the tubular penile urethra. The result of this in humans is the presence of an abnormal ventral urethral meatus, incomplete formation of the prepuce and an abnormal penile curvature.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

Development of the male external genitalia, which occurs in the fetal period of development, is androgen dependent and involves epithelial-mesenchymal interactions. Because of these interactions, which are very similar to limb development, research into the development of genital tubercle has utilised similar methods for both processes. A minority of hypospadias cases are a result of the androgenic pathways being impaired and causing this congenital defect. The cell-cell interactions that allow for the development of the male external genitalia are mediated by a broad range of signaling molecules and growth factors such as fibroblast growth factors (FGFs), Sonic hedgehog (SHH) and bone morphogenetic proteins (BMPs). Such signaling and growth factors are downstream of androgen receptor signaling and an understanding of the mechanisms that underlie normal penile development during the fetal period, will lead to a deeper understanding of the aetiology of hypospadias.<ref name=PMID14641326><pubmed>14641326</pubmed></ref>

----

'''Paracetamol, aspirin, and indomethacin induce endocrine disturbances in the human fetal testis capable of interfering with testicular descent.''' '''(2013)'''<ref name=PMID24030937><pubmed>24030937</pubmed></ref>

<pubmed>24030937</pubmed>

The differentiation of the gonads into male or female begins around week 6-8 and is linked to the sex-determining region of the Y chromosome. The formation and descent of the testis is determined by a number of hormones which are:
*Anti-mullerian hormone (AMH) - produced by sertoli cells, which acts on the mullerian ducts.
*Testosterone - produced by the fetal leydig cells, which ensures differentiation of wolffian ducts as well as the terminal phase of descent of the testis.
*Insulin like factor 3 (INSL3) - is produced by the differentiated fetal leydig cells, which are involved in the transabdominal phase of descent.
*Prostaglandins are also believed to be involved in the differentiation of the male genital tract and testis.

The development of the male reproductive system requires the action of different hormones and is highly susceptible for development to be altered due to endocrine disruptions.

Cryptorchidism is the failure of descent of the testis and is the most common congenital malformation in males.
Non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol are some of the most widely used drugs used. These drugs have recently been identified as potential endocrine disruptors (ED) in humans. A number of epidemiological studies have reported that exposure to NSAIDs and analgesics during pregnancy showed an increased risk of cryptorchidism.

This study used 62 fetuses from the first trimester between 7-12 weeks in gestation (GW) from pregnant women who obtained an abortion legally and with were given information and verbal consent was obtained according to national guidelines. The terminations were not motivated by abnormalities. The testes were cut in approximately 1mm3 pieces and drugs were used in the same concentration compared to recommended dosages in the body. The drugs used were paracetamol, aspirin, indomethacin, ketoconazole (antifungal).

Testicular cells were counted using histology and image analysis and the hormones were assayed in the medium.
The results showed no changes in the architecture of the testis with the analgesic treatment whereas the ketoconazole caused the boundaries of the testis cords to become unrecognisable. The analgesics did not significantly modify the number of germ cells or sertoli cells.
Ketoconazole reduced testosterone levels in contrast to indomethacin, which stimulated testosterone production. Paracetamol had no significant effect on testosterone while aspirin produced a dose response relationship with an increase in testosterone after 72 hours in the youngest fetuses (8-9.86 GW) but not the older testes (10-12GW). None of the analgesics significantly affect the number of interstitial cells.

The results showed a consistent trend for lower INSL3 production after 48-72 hours of exposure to mild analgesics and ketoconazole. This was the first study to measure direct production of INSL3 by the testis.
Aspirin strongly stimulated AMH production, whereas as paracetamol and indomethacin increased production but not significantly. The analgesics did not significantly alter the sertoli cells and Ketoconazole significantly inhibited AMH production. Aspirin and paracetamol showed significant inhibition of Prostaglandin E2 production while indomethacin had no effect.

In conclusion, the study shows that painkillers have a direct effect on various hormones, which are crucial for endocrine function and development of the human testis. The study shows that there is a direct effect with the dosages which are currently found with most medications.

----

'''The Effect of Dihydrotestosterone Exposure During or Prior to the Masculinisation Programming Window on Reproductive Development in Male and Female Rats (2012)'''<ref name=PMID22248293><pubmed>22248293</pubmed></ref>

<pubmed>22248293</pubmed>

[[File:Schematic representation of the various treatment windows and experimental design..jpg|400px|thumb|left|Schematic representation of the various treatment windows and experimental design]]

Whilst it is evident that some of the most common reproductive abnormalities in males occurs within the fetal stage, it is believed that disorders that affect young men later in life (such as low sperm count or testicular cancer) could also occur within this stage. Previous studies have demonstrated a critical period (masculinisation programming window - MPW) during fetal development that must transpire in order for masculinisation of the male fetus to occur. If the androgenic pathways do not occur in the correct manner, testicular dysgenisis (TD) may result. Although androgen production can be difficult to measure in humans, it can be measured by examining the anogenital distance, which is shorter in females versus males.

Therefore, it can be established that the MPW is important in setting up normal male development and it is triggered to open by either the presence or absence of both androgens and their receptors. The aim of the present study was to investigate whether or not availability of excess androgens available to their receptors before or during the MPW could increase masculinisation and therefore, development of male and female rats.

This study uses the highly potent dihydrotestosterone (DHT) androgen in comparison to other studies, which used testosterone, with the potential to convert to oestradiol and cause pregnancy disorders. The study used pregnant Wister rats and exposed them to DHT in two time frames: either before the MPW or during it. Relevant tissues were then extracted from the animals and certain procedures such as spectrophotometry, immunohistochemistry for AR proteins and RNA analysis were used to measure masculinisation and obtain results.

Results showed that exposing male fetuses to a dosage of DHT that would result in masculinisation of the female fetus had no effect on the male. More specifically, the results indicated that exposure to DHT before or during the MPW did not stimulate any male reproductive development, and female masculinisation may begin much prior to the MPW. The results also support previous evidence demonstrating the potential for testosterone and oestrogen treatment to cause adverse pregnancy effects.

Overall, the study concludes that genital development in male rats is not enhanced by DHT exposure before or during the MPW and that sensitivity of the female fetus to androgens can not only be refined to occurring during the MPW but also prior to. This has implications in that the female fetus is more susceptible to androgens and masculinisation of the genital system much before the male is, therefore suggesting a wider window.

====Female====
----

'''Female External Genitalia on Fetal Magnetic Resonance Imaging (2011)'''<ref name=PMID21584884><pubmed>21584884</pubmed></ref>

<pubmed>21584884</pubmed>

In the past, magnetic resonance imaging (MRI) has been used in conjunction with ultrasound to diagnose prenatal complications, including urogenital abnormalities. However, no previous research yielded any data regarding normal development of female external genitalia using MRI. As a result, this study investigated labial growth and correlated it with gestational age using prenatal MRI.

This study included fetal MRI results from 197 female fetuses of Caucasian background, with either normal anatomy or minor congenital abnormalities. Fetuses with major congenital abnormalities, especially with urogenital abnormalities were omitted from the study. The MRI results were used to confirm if suspected anomalies during ultrasound screening were correct. Axial and coronal images of the fetus’ lower body were used to visualise the external female genitalia, including labia and clitoris and statistical analyses were performed on all MR images.

Results showed a linear relationship between bilabial diameter and gestational age, and the morphology on the MRI showed a statistically significant difference between the 20-23 weeks age group and the rest (24-36 weeks) in the visual differentiation of the clitoris and the labial structures. Between 20-23 weeks, differentiation of the clitoris from the labia was not possible. Similar to what can be identified in ultrasounds, from 24 weeks onwards, in 12% of fetuses the clitoris and labia could be differentiated as 3-5 protuberances emerging from the pelvis, with the clitoris lying in the midline.

The results are important as they demonstrate the MRI’s potential to be used in adjunct to ultrasound in order to assist in the diagnosis of certain genital abnormalities, such as hypospadias or micropenis. This is necessary as such conditions can mimic female external genitalia and so hypospadias especially should be determined on the basis of parallel labial lines and not exclusively on the direction of the genital tubercle.<ref name=PMID18431748><pubmed>18431748</pubmed></ref>

In conclusion, this study examines the morphological development of the female external genitalia in utero using MRI, proving its effectiveness as a visualiser of the female phenotype and diagnosis of genital abnormalities, and should be used in conjunction with ultrasound.

----

'''In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (2011)'''<ref name=PMID16585961><pubmed>16585961</pubmed></ref>

<pubmed>21584884</pubmed>

As sexual differentiation of the external genitalia is an event that occurs within the fetal period, it is highly important to maintain the correct intrauterine environment in terms of hormonal content. In humans, differentiation and growth of the external genitalia is triggered by the release of androgens from the fetal testis such as dihydrotestosterone. Exposure of the female fetus to these androgens results in the female developing more male sexual characteristics at birth. This results from congenital adrenal hyperplasia, due to a deficiency of cytochrome P450 21-hydroxylase (CYP21), an enzyme involved in the biosynthesis of cortisol.

A deficiency of CYP21 results in a decrease in cortisol levels, and this is believed to alleviate negative feedback at the fetal anterior pituitary. As a result, increased adrenocorticotropic hormone (ACTH) shifts steroid precursor formation towards androgen biosynthesis and therefore a balance between cortisol biosynthesis and androgen production is important for normal female external genitalia development. Therefore, this article illustrates the potential of utilising early cortisol biosynthesis to uphold normal female sexual development.

The study used gas chromatography and mass spectrometry to observe a 9-18 fold increase in cortisol levels within the adrenal gland during the first trimester. The capacity of the adrenal gland in the fetus to secrete androgens was also determined using assays.

By the time differentiation of the external genitalia occurs in the second trimester, the female fetus is well protected by high levels of placental aromatase enzymes, which convert androgens to oestrogens. Also, in order to prevent virilisation in CYP21 deficiency, dexamethasone needs to be administered at week 6.

----

'''Fibrillin-3 in the Fetal Ovary: Can it Contribute to Polycystic Ovary Syndrome? (2012)'''<ref name=PMID23585338><pubmed>23585338</pubmed></ref>

<pubmed>23585338</pubmed>

Fibrillin-3 contributes to microfibril formation within the extracellular matrix of many mammals and is predominantly expressed during fetal life<ref name=PMID20970500><pubmed>20970500</pubmed></ref>. The ovary continuously remodels its stroma in order to accommodate the constantly growing follicles from fetal life through to adult life, resulting in a continuously changing extracellular matrix, and therefore, fibrillin-3. Recent studies have discovered a linkage between an allele of the fibrillin-3 gene and polycystic ovary syndrome (PCOS), where hyperandrogenic interactions result in an enlarged and hyperstromal ovary with follicles that fail to mature and get released.

This study obtained 29 samples of ovarian tissue from humans in first or second trimester fetuses and 6 non-PCOS adults. Fetal bovine ovaries were also obtained. mRNA expression analyses were performed, as well as PCR and indirect immunofluorescence immunochemistry.

Results showed that in both human and bovine ovaries, fibrillin-3 mRNA is mostly expressed during the first trimester, with little to none being expressed in the adult ovaries. Localised expression of fibrillin-3 surrounding primordial and primary follicles results in fetal oocyte and adult follicle expansion within the stroma, as the ECM remodeling is necessary to support the growth of these follicles. Therefore, the fibrillin-3 gene in PCOS women displays potential for altering fetal ovarian follicle development, and since it is expressed in ECM throughout the fetus, it may result in altered development in non-ovarian organ systems in human fetuses.

In conclusion, the study suggests that since the stroma within the ovaries is hyper developed in PCOS women, different alleles of the same fibrillin-3 gene could be expressed within the fetal ovary to overcommit the polycystic ovary to follicular growth that is unlikely to mature into preovulatory follicles.

----

'''Expression of miRNAs in Ovine Fetal Gonads: Potential Role in Gonadal Differentiation (2011)'''<ref name=PMID21223560><pubmed>21223560</pubmed></ref>

<pubmed>21223560</pubmed>

Genotype of sex is determined at the time of fertilisation, where a sperm carrying X or Y genetic material will fertilise an XX ova. This genotype that results then determines whether the genital ridge in the embryonic period will develop into the fetal testis (XY) or fetal ovaries (XX). The pathway involving testicular development includes a fine balance between genes that promote testis development and simultaneously genes that prevent ovarian development <ref name=PMID19027189><pubmed>19027189</pubmed></ref>

Some critical genes involved in the testicular and ovarian pathways include:
*SRY gene (sex-determining region of the Y-chromosome) <ref name=PMID2247149><pubmed>2247149</pubmed></ref>
*Rspol gene (R-spondin homolog)<ref name=PMID18250098><pubmed>18250098</pubmed></ref>
*Wnt4 – wongless-related MMTV integration site 4)<ref name=PMID18250097><pubmed>18250097</pubmed></ref>
*Beta-catenin<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Such genes are expressed in the support cells of the fetal gonads, for example, the Sertoli cells in the testis and the granulosa cells in the ovary.<ref name=PMID18250098><pubmed>18250098</pubmed></ref>

Small non-coding RNA molecules, called miRNAs are RNAs that regulate gene expression and function within many different tissue types. Whilst studies have shown that miRNAs are important for growth and development of the gonads, none have yet indicated which miRNAs.

Not much is known regarding the expression of miRNAs during fetal genital development in mammals and the purpose of this study was to identify this expression of miRNAs using the ovine as a model. Expression levels were examined and the importance of such research is to provide further understanding of human genital development on a genetic level, as well as the reproductive development of ovine, which may have economical implications as livestock.

This study used sheep breeding methods and collected fetal gonads, which then underwent PCR genotyping. RNA was isolated, and miRNAs were treated with reverse transcriptase and then hybridised. These techniques were all used to detect expression levels of the relevant genes.

From the study, it is evident that miRNAs are indeed present during fetal genital development in sheep. It is believed that miRNAs are important regulators of gene expression and function and based upon the results, the genes Let7 and miR-22 regulate oestrogen signaling during fetal genital development. Further, miR-22 may be needed for suppression of the oestrogen-signaling pathway during fetal development of the testes, as localisation of the gene in the testicular cords suggested that Sertoli cell development required such suppression of the oestrogen-signaling pathway.

----

{| class="wikitable mw-collapsible mw-collapsed"
! '''Other current research findings and interesting reads:'''
|-
| * A recent study investigated the ability of in vitro cultures of female fetal mouse gonads to subsequently develop in vivo. It demonstrated that premeiotic germ cells in fetal gonads possessed the capability to develop into mature oocytes using this method. <ref name=PMID19379463><pubmed>19379463</pubmed></ref>Additionally, the study showed that the longer a culture of fetal gonads was kept (>14 days), follicular and development and oocyte growth in vivo was affected, as well as the maturation of the oocytes in vitro following transplantation into kidney capsules (the capsules are an ectopic site, however have all the necessary conditions for growth of the oocytes). <ref name=PMID8882299><pubmed>8882299</pubmed></ref>

<pubmed>21584884</pubmed>
<pubmed>18367374</pubmed>
<pubmed>15086026</pubmed>
<pubmed>14641326</pubmed>
<pubmed>11684660</pubmed>
<pubmed>22127979</pubmed>
<pubmed>24631756</pubmed>
<pubmed>23192465</pubmed>
|}

----

==Historic Finding==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==Abnormalities==

[https://embryology.med.unsw.edu.au/embryology/index.php/2014_Group_Project_9]

==References==
<references/>

File:Clitoris.jpg

2014-10-23T11:14:53Z

Z3417753: ===Stages in the Development of the External Sexual Organs in the Male and Female=== * Drawn from the Ecker-Ziegler models. ====References==== Image URL: http://commons.wikimedia.org/wiki/File:Stages_in_the_development_of_the_external_sexual_organs_i...

===Stages in the Development of the External Sexual Organs in the Male and Female===
* Drawn from the Ecker-Ziegler models.

====References====

Image URL: http://commons.wikimedia.org/wiki/File:Stages_in_the_development_of_the_external_sexual_organs_in_the_female_(from_Gray1119).png

Book reference: Gray, Henry. Anatomy of the Human Body. Philadelphia: Lea & Febiger, 1918.

Original Author: Gray's Anatomy

====Copyright====

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