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'''Artificial Reproductive Technologies (ART)''' | '''Artificial Reproductive Technologies (ART)''' | ||
Ass. Prof. Gilchrist--> womens health and IVF | Ass. Prof. Gilchrist--> womens health and IVF | ||
Lab 2 | |||
'''Lab 2''' | |||
*IVF partnership between scientists and doctors -- management of patient and their needs as well as research in labs | *IVF partnership between scientists and doctors -- management of patient and their needs as well as research in labs | ||
Line 30: | Line 31: | ||
* Very controversial at the time | * Very controversial at the time | ||
* 10 embryos--> 5 are carriers; 2.5 are affected and 2.5 are unaffected | * 10 embryos--> 5 are carriers; 2.5 are affected and 2.5 are unaffected | ||
* 1 blastomere (cell) is extracted and tested for the disease (once removed and tested the blastocyst is destroyed--- no huge implications for the embryo) | * 1 blastomere (cell) is extracted and tested for the disease (once removed and tested the blastocyst is destroyed--- no huge implications for the embryo) | ||
* it is possible to sequence an entire genome from the one cell (predicting higher propencities for strokes or CVD -- Illegal in Australia) | |||
* process= Comparactive Genomic hybridisation) | |||
* eg's Aneuploidy, trisomy 21, translocations, single gene defects | |||
* in AUS only legal if the offspring will be affected by a devastating disease eg Trisomy 21 --> no sex selection, unless for a sex linked disease | |||
Sperm Injection = 1992 | Sperm Injection = 1992 | ||
Line 55: | Line 59: | ||
Ovarian reserve testing = 2005 | Ovarian reserve testing = 2005 | ||
* the ability to approximate the number of oocytes a woman has left | * the ability to approximate the number of oocytes a woman has left-- blood test to measure AMH and AFC (ultrasound) can be used to estimate the Ovarian reserve | ||
* important due to the increase in average maternal age | * important due to the increase in average maternal age | ||
* womans fertility depletes almost completely at 40 (due to the loss of primordial follicles) | |||
* can sell eggs in America-- clincaly and comercialy relavant in the last 10 years | |||
* new form is being developed that tests for a hormone released directly from the oocyte | |||
* particularly relevant for young women with cancer or a carrier of breast cancer gene (allowing women to preserve ovarian reserve prior to treatments that can deplete/destroy the ovary) | |||
OOcyte Freezing, social freezing =2008 | OOcyte Freezing, social freezing =2008 | ||
* ability to delay child bearing | * ability to delay child bearing years | ||
* in conjunction with ORT | |||
Whole genome screening of embryos= 2013 | Whole genome screening of embryos= 2013 | ||
Line 97: | Line 107: | ||
6. Transfer of Embryo | 6. Transfer of Embryo | ||
Ovarian in Vitro Maturation-- collection of follicles at a less mature stage (cultured for ~24 hours before going to IVF) | |||
* follicle arrested at P1 | |||
* mature as normally once in a solution of LH (complex medium, protein, FSH) | |||
* differences to IVF--- there are no hormones; the oocyte is just picked up and then cultured in vitro | |||
'''Lab 3''' | |||
Transition from week 3 trilaminar embryo into early development (weeks 3 & 4) | |||
WEEK3 | |||
* Crown-rump measurement (~3cm) --> not an actual measurement of the embryo due to shrinkage (a result of fixation) and different fixatives and variables result in different degrees of shrinkage. | |||
* Major events occurring in week 3 | |||
* amniotic sac on the dorsal side of the embryo--> sac lined with ectoderm (embryonic and epithelium lining cavity) | |||
* surface of amniotic sac --> extra embryonic mesoderm-- contributing to fetal membranes and placenta | |||
* Corionic cavity lined with extra-embryonic mesoderm | |||
* endoderm forms the ventral side of the embryo | |||
* Cranial end--> cardiogenic region; differentiating within mesoderm | |||
* folding of embryo ventrally bringing connecting stalk to the ventral side of embryo | |||
* umbilicus --> site of connection of chord, yolk stalk etc | |||
* sac covering embryo-- therefore the remainder of embryonic and fetal development occurs within the sac | |||
* oral membrane -- primitive mouth area | |||
* folding of the yolk sac into the embryo forms the fore, mid and hind gut | |||
* beneath heart-- transverse septum (mesoderm) contributing to liver development and the central tendon of the diaphragm | |||
* fold of endoderm extending into the connecting stalk -- blind ended tube (hollow)-- allantois (remnant of evolution or an unidentified specialised function) forming the superior end of the urinary bladder | |||
* Midgut connected by yolk stalk to the yolk sac (outside the embryo) | |||
* blood vessels that form on the yolk sac are important to the development of portal vasculature | |||
* Head paraxial mesoderm segments into somites (44-48 pairs) in a rostro-caudial direction (from head to bottom) | |||
* Intermediate Mesoderm forms 3 pairs of kidneys (pronephros, mesonephros and metanephros) | |||
* Lateral plate mesoderm --> horse shoe cavity around the cranial end separting mesoderm into somatic and splanchnic mesoderm (forming heart) | |||
* horse shoe cavity forms the 3 body cavities (pericardial, pleural and peritoneal) | |||
WEEK4 | |||
* embryo has different stem cells in different locations eg GIT, skin etc | |||
* if they grow out of control-- form tumors and cancers | |||
* totipotential eg blastomere--> undifferentiated cells that can form many tissues or cells | |||
* Primary vesicles (3) form and these then subdivide into the 5 secondary vesicles | |||
* GIT nervous system--> innervation of peristalsis (regulated by enteric ns) | |||
* Pharyngeal Arches ( 1-4 +6 --> 1 is largest and 4+6 fuse) | |||
* arch 5 if it occurs is a transient structure--> present in non-humans | |||
* each sensory system has its own placode that is associated with it eg otic placode forming the entire inner ear; optic placode forms the lens of the eye | |||
* limb buds initially formed (upper) on either side of the heart | |||
* limb buds are ecto and endoderm | |||
ABNORMALITIES | |||
* Neural rube defects | |||
* defective fusion of the neural folds--> dysraphism | |||
* folate in flour | |||
* still a slight occurance of these abnormalities because there are many ways to generate these abnormalities independent of folate eg clef palate | |||
'''Lab 4''' | |||
Blood and early development of the Cardio-vascular system | |||
* fetal cells necessary to make placenta only available at implantation | |||
* blastocyst--> major player on placental development (trophoblast- forming syncitiotrophoblast and cytotrophoblasts) | |||
* syn-- outside, multi nucleated, synthesis hCG (secreted locally and taken up by maternal blood at lacunae) | |||
* cyt-- inside, single nucleus | |||
* luteum is yellow when producing hormone and albicans is white when not producing hormone | |||
* luteum produces estrogen and progesterone | |||
* progesterone suspends uterus lining in the secretory phase and therefore preserving implantation | |||
* placenta takes over from luteum--> endocrine role | |||
* extra-villus cytotrophoblast--> surround ends of placental vessels and important in maintaining placental blood flow | |||
* decidualisation--> important in contributing maternal component of placenta | |||
* three extra-embryonic chambers -- amniotic, yolk sac and chorionic cavity (largest space outside of embryo, lined with extra-embryonic mesoderm) | |||
* conceptus-- embryo and extra-embryonic cells--> all cellular products | |||
* when conceptus is divided (wk2-4)- uterine side vs. maternal blood side | |||
* uterine side--> not richly vascularised and small amount of villus development | |||
* Maternal blood side --> access nutrients, strong growth factor drive, large amount of villus development and richly vascularised | |||
* chorion laeve--> smooth, not many villi | |||
* chorion frondosum --> leafy chorion; side where placenta will form | |||
* desudua vasalis-- maternal contribution to placenta | |||
* capsularis -- lie over chorion laeva | |||
* parietalis | |||
* three stages of villi development--primary, secondary and tertiary | |||
* primary villi--> trophoblastic shell cells | |||
* secondary villi--> extra-embryonic mesoderm grows into villi | |||
* tertiary villi--> formation of blood vessels | |||
* fetal infections passed through the placenta eg malaria -- a number of immune structures to pass through | |||
* abnormal placentation-- in lobes or cord not centric can lead to impaired embryonic development | |||
* embryonic contribution to the placenta-- cytotrophoblast layer, trophoblast column (connecting site for villi), anchoring villi (attaching to maternal decidua), syncitiotrophoblast (as islands and on the outside of cytotrophoblasts) | |||
* cytotrophoblasts-- transient, from cuboidal to squamous and can degenerate | |||
* no direct contact between maternal and fetal blood | |||
* during pregnancy-- arterial/venous shunt formed in uterine wall (homogenous oxygen level within lacunae) | |||
* Eclampsia-- hypertension resulting in the decrease of oxygen exchange | |||
* accreta--> abnormal adherence and the absence of basalis |
Latest revision as of 13:27, 28 August 2015
Artificial Reproductive Technologies (ART) Ass. Prof. Gilchrist--> womens health and IVF
Lab 2
- IVF partnership between scientists and doctors -- management of patient and their needs as well as research in labs
- Growing industry--> IVF and also research and technical sides
- ~5 million IVF offspring living today--> arguably greatest medical success stories of our time = an emotional process for couples
- AUstralia has and continues to be a pioneer in this areas
Just under a million couples each year. 1/6 Australian couples are treated for infertility--> in 2010 ~3.3% of babies were IVF. IVF hormones cost the PBS ~$100 million/year--> movement out of the private sector. Subsidized by Medicare (a large portion covered- particularly the drugs and ~six rounds of IVF) therefore it is not as expensive in aus.
Marriage of Science Research and Medicine;
Wolrds first IVF baby born in 1978-- Louise Brown-- very controversial- born in cambridge BOB EDWARDS won the Nobel Prize for Medicine for his role in the production of IVF technologies--- extremely controversial (unhealthy children)
Donor Sperm= 1960; poor quality/ no sperm
- artificial insemination
Ovulation induction= 1960
- failure of follicle to ovulate
- One injection of hCG to induce ovulation
Donor Egg= no/ poor quality eggs -->1984
Pre-implantation genetic diagnosis= 1988
- diagnosing genetic abnormalities
- ability to remove cystic fibrosis (single gene following Mendilian ratio- very easy to detect) from a family--> are we removing evolution in a Darwinian sense?
- Very controversial at the time
- 10 embryos--> 5 are carriers; 2.5 are affected and 2.5 are unaffected
- 1 blastomere (cell) is extracted and tested for the disease (once removed and tested the blastocyst is destroyed--- no huge implications for the embryo)
- it is possible to sequence an entire genome from the one cell (predicting higher propencities for strokes or CVD -- Illegal in Australia)
- process= Comparactive Genomic hybridisation)
- eg's Aneuploidy, trisomy 21, translocations, single gene defects
- in AUS only legal if the offspring will be affected by a devastating disease eg Trisomy 21 --> no sex selection, unless for a sex linked disease
Sperm Injection = 1992
- instead of bathing egg in sperm
- 70-80% of all IVF are now CSI
- sperm is injected into the oocyte
Blastocyst Development- 1994
- decades of research of metabolic need of pre-implanatation embryos
Sex selection= 1998
- capacity to sort X or Y bearing chromosome sperm and being able to inseminate based on chromosome
- production of only male or female embryos
- only allowed in aus. based on sex linked diseases
- China-- very high pressure for sex selection= single child policy and a tradition for male children (it is not legal but it does happen)
Dolly= 2000
- stem cells, cloning and genetic transfer
- first demonstration of being able to clone an animal
- therefore you can easily clone a human-- the same technology therefore very controversial
- gvmt bodies and regulatory bodies preventing the cloning of humans-- banned in most/all countries (laws restrict drs actions--> no dr is going to break the law)
- problem is-- certain aspects of technology leads to health problems of the embryo
Ovarian reserve testing = 2005
- the ability to approximate the number of oocytes a woman has left-- blood test to measure AMH and AFC (ultrasound) can be used to estimate the Ovarian reserve
- important due to the increase in average maternal age
- womans fertility depletes almost completely at 40 (due to the loss of primordial follicles)
- can sell eggs in America-- clincaly and comercialy relavant in the last 10 years
- new form is being developed that tests for a hormone released directly from the oocyte
- particularly relevant for young women with cancer or a carrier of breast cancer gene (allowing women to preserve ovarian reserve prior to treatments that can deplete/destroy the ovary)
OOcyte Freezing, social freezing =2008
- ability to delay child bearing years
- in conjunction with ORT
Whole genome screening of embryos= 2013
- shanghai
- examining all the genes in an embryo
Recently= mitochondrial transfer-- three types of genetic material in offspring
- Built on the back of 20 years of animal research
Bovine industry--> freezing of embryos
- allowing freezing of embryos in liquid nitrogen
IVF
1.Ovarian Hyperstimulation--> to generate multiple follicles and oocytes-- stems from animal research
- normally only one follicle reaches ovulation
- ~30 follicles do not make it to ovulation but will also be lost
- Inject patient with FSH (pituitary) promoting the growth of follicles--> tightly controlled by negative feedback of progesterone
- Increase FSH (~10 injections and pituitary down regulation stopping the negative feedback) resulting in ~20 follicles rather than 1
- Oestrogen produced by follicles granulosa cells-- 20 x more oestrogen produced with the ovulation of 20 follicles resulting in Ovarian Hyperstimulation Syndrome
- Large dose of hCG-- analog to LH-- stimulating maturation of oocyte in the follicle and allowing them to be collected
- LH normally stimulates ovulation
2. Oocyte Pick-up--> originally by laproscopy (through the abdominal wall); ~20 years to trans-vaginal oocyte recovery (ultrasound probe with needle at end inserted into vagina and through the ovary wall-- out-patient procedure, usually under light sedation)
3. Sperm retrieval--> ~90% via masturbation; some by surgery ( problems with ejaculation or low numbers of sperm-- from epidydimis or testes)
- capacitation-- cultured for a few hours
4. IVF
- occurs in a dish
- ICSI-- sperm injection into the oocyte via two glass needles --> guarantees that the sperm gets into the oocyte (practically curing male infertility over night)
- very technical procedure- lots of patients and a growing area of ART
5. Embryo culture
- With 8 cells the embryo can be frozen and transfered into the patient (day 3)
- Blastocyst growth is more common to be transferred into the patient-- higher likelihood for success
6. Transfer of Embryo
Ovarian in Vitro Maturation-- collection of follicles at a less mature stage (cultured for ~24 hours before going to IVF)
- follicle arrested at P1
- mature as normally once in a solution of LH (complex medium, protein, FSH)
- differences to IVF--- there are no hormones; the oocyte is just picked up and then cultured in vitro
Lab 3
Transition from week 3 trilaminar embryo into early development (weeks 3 & 4)
WEEK3
- Crown-rump measurement (~3cm) --> not an actual measurement of the embryo due to shrinkage (a result of fixation) and different fixatives and variables result in different degrees of shrinkage.
- Major events occurring in week 3
- amniotic sac on the dorsal side of the embryo--> sac lined with ectoderm (embryonic and epithelium lining cavity)
- surface of amniotic sac --> extra embryonic mesoderm-- contributing to fetal membranes and placenta
- Corionic cavity lined with extra-embryonic mesoderm
- endoderm forms the ventral side of the embryo
- Cranial end--> cardiogenic region; differentiating within mesoderm
- folding of embryo ventrally bringing connecting stalk to the ventral side of embryo
- umbilicus --> site of connection of chord, yolk stalk etc
- sac covering embryo-- therefore the remainder of embryonic and fetal development occurs within the sac
- oral membrane -- primitive mouth area
- folding of the yolk sac into the embryo forms the fore, mid and hind gut
- beneath heart-- transverse septum (mesoderm) contributing to liver development and the central tendon of the diaphragm
- fold of endoderm extending into the connecting stalk -- blind ended tube (hollow)-- allantois (remnant of evolution or an unidentified specialised function) forming the superior end of the urinary bladder
- Midgut connected by yolk stalk to the yolk sac (outside the embryo)
- blood vessels that form on the yolk sac are important to the development of portal vasculature
- Head paraxial mesoderm segments into somites (44-48 pairs) in a rostro-caudial direction (from head to bottom)
- Intermediate Mesoderm forms 3 pairs of kidneys (pronephros, mesonephros and metanephros)
- Lateral plate mesoderm --> horse shoe cavity around the cranial end separting mesoderm into somatic and splanchnic mesoderm (forming heart)
- horse shoe cavity forms the 3 body cavities (pericardial, pleural and peritoneal)
WEEK4
- embryo has different stem cells in different locations eg GIT, skin etc
- if they grow out of control-- form tumors and cancers
- totipotential eg blastomere--> undifferentiated cells that can form many tissues or cells
- Primary vesicles (3) form and these then subdivide into the 5 secondary vesicles
- GIT nervous system--> innervation of peristalsis (regulated by enteric ns)
- Pharyngeal Arches ( 1-4 +6 --> 1 is largest and 4+6 fuse)
- arch 5 if it occurs is a transient structure--> present in non-humans
- each sensory system has its own placode that is associated with it eg otic placode forming the entire inner ear; optic placode forms the lens of the eye
- limb buds initially formed (upper) on either side of the heart
- limb buds are ecto and endoderm
ABNORMALITIES
- Neural rube defects
- defective fusion of the neural folds--> dysraphism
- folate in flour
- still a slight occurance of these abnormalities because there are many ways to generate these abnormalities independent of folate eg clef palate
Lab 4
Blood and early development of the Cardio-vascular system
- fetal cells necessary to make placenta only available at implantation
- blastocyst--> major player on placental development (trophoblast- forming syncitiotrophoblast and cytotrophoblasts)
- syn-- outside, multi nucleated, synthesis hCG (secreted locally and taken up by maternal blood at lacunae)
- cyt-- inside, single nucleus
- luteum is yellow when producing hormone and albicans is white when not producing hormone
- luteum produces estrogen and progesterone
- progesterone suspends uterus lining in the secretory phase and therefore preserving implantation
- placenta takes over from luteum--> endocrine role
- extra-villus cytotrophoblast--> surround ends of placental vessels and important in maintaining placental blood flow
- decidualisation--> important in contributing maternal component of placenta
- three extra-embryonic chambers -- amniotic, yolk sac and chorionic cavity (largest space outside of embryo, lined with extra-embryonic mesoderm)
- conceptus-- embryo and extra-embryonic cells--> all cellular products
- when conceptus is divided (wk2-4)- uterine side vs. maternal blood side
- uterine side--> not richly vascularised and small amount of villus development
- Maternal blood side --> access nutrients, strong growth factor drive, large amount of villus development and richly vascularised
- chorion laeve--> smooth, not many villi
- chorion frondosum --> leafy chorion; side where placenta will form
- desudua vasalis-- maternal contribution to placenta
- capsularis -- lie over chorion laeva
- parietalis
- three stages of villi development--primary, secondary and tertiary
- primary villi--> trophoblastic shell cells
- secondary villi--> extra-embryonic mesoderm grows into villi
- tertiary villi--> formation of blood vessels
- fetal infections passed through the placenta eg malaria -- a number of immune structures to pass through
- abnormal placentation-- in lobes or cord not centric can lead to impaired embryonic development
- embryonic contribution to the placenta-- cytotrophoblast layer, trophoblast column (connecting site for villi), anchoring villi (attaching to maternal decidua), syncitiotrophoblast (as islands and on the outside of cytotrophoblasts)
- cytotrophoblasts-- transient, from cuboidal to squamous and can degenerate
- no direct contact between maternal and fetal blood
- during pregnancy-- arterial/venous shunt formed in uterine wall (homogenous oxygen level within lacunae)
- Eclampsia-- hypertension resulting in the decrease of oxygen exchange
- accreta--> abnormal adherence and the absence of basalis