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''Creating a Karyotype''
''Creating a Karyotype''
[[Image:Trisomy21female.jpg|thumb|alignment|left|180px|A karyotype of a human female showing the standard layout of chromosomes, and the abnormality of trisomy of chromosome 21 resulting in Down syndrome.]]
[[Image:Trisomy21female.jpg|thumb|alignment|left|200px|A karyotype of a human female showing the standard layout of chromosomes, and the abnormality of trisomy of chromosome 21 resulting in Down syndrome.]]




* The standard way of observing the stained chromosomes is by creating a karyotype.<ref name="PMID5273905 "/> A karyotype is a photograph taken of the chromosomes arranged from largest to smallest non-sex chromosomes (autosomal) numbered 1 to 22, with the 2 sex chromosomes placed at the end. This allows analysis to be done efficiently and any structural abnormalities to be clearly identified.
* The standard way of observing the stained chromosomes is by creating a karyotype.<ref name="PMID5273905 "/> A karyotype is a photograph taken of the chromosomes arranged from largest to smallest non-sex chromosomes (autosomal) numbered 1 to 22, with the 2 sex chromosomes placed at the end. This allows analysis to be done efficiently and any structural abnormalities to be clearly identified.
* Structural abnormalities that may be indicative of a genetic disorder include chromosome deletions, duplications, translocations and inversions.
* Structural abnormalities that may be indicative of a genetic disorder include chromosome deletions, duplications, translocations and inversions.


==Risks==
==Risks==

Revision as of 13:33, 11 September 2010

Amniocentesis

Introduction

Developing human embryo showing the surrounding amniotic fluid from which the sample is taken.

Amniocentesis is the process by which a thin needle is inserted through a womens abdomen into her womb, a sample of the amniotic fluid surrounding her growing fetus is taken out and analysed to acquire information about the baby's health.


This procedure is an important development in the technologies of prenatal diagnostic techniques as it allows parents an insight into possible diseases and complications that their baby might develop before birth at an early stage of its development.


Although it is a very useful technique in diagnosing up to several hundred fetal complications there are also many risks associated with this invasive method of extraction of fetal cells as well as ethical issues which will also be explored. A brief history of the development of amniocentesis will be explained, as well as the key disorders which may be detected using it. Lastly we will explore the accuracy of this test against other prenatal diagnostic techniques and highlight current research being conducted.

Historic Background

The removal of amniotic fluid using a needle has been recorded as early as 1930 by Menees, however it was not until 20 years later that the process was actually used in a diagnostic sense. The development of safe and effective techniques of the procedure took many years to evolve, including the necessary technologies to come to par such as ultrasound and chromosome mapping that are key features in ensuring safety and effectiveness in diagnosing the fetus.

This progress could not have been achieved without the work of several key scientists:

Douglas Bevis

Bevis's study of amniocentesis and hemolytic disease was considered a landmark event making the procedure wide spread and launching more interest and research into its possibilities. In February 1952 he published his study in the Lancet, "The Antenatal Prediction of a Hemolytic Disease of the Newborn" which was a revolutionary article at the time. Bevis experimented with the mixing of Rh+ and Rh- blood and what occurs when there is a clash between the mothers Rh factor and her fetus's Rh factor. This results in hemolytic disease, a disease in newborns where the mothers antibodies attack the fetal red blood cells when the antibodies cross the placenta where they are supposed to strengthen the babies immune system.[1] The rhesus factor is present and able to be identified in the fetus from the sixth week of development onwards.[2] The possibility of diagnosing this disorder prenatally was a critical discovery and paved the way for the discovery of hundreds of other disorders which are able to be prenatally diagnosed.

Murray Barr and Ewart Bartram

The presence of the Barr body in female cells.

In the late 1940 's these two scientists began working together studying human sex cells. They discovered in 1949 that the sex of a fetus could be found by identifying an extra chromatin body which lies at the periphery of only female cells, named the Barr body, which is visible under the microscope. The significance of this in relation to amniocentesis as a diagnostic technique is that the possibility of sex linked diseases such as hemophilia, a blood clotting disorder, could be found out prenatally. Murray Barr explains the presence of the chromatin body in his paper Prenatal Sex Determination written in 1956[3], however he comments that puncturing the abdomen and uterus of the mother is not worth the associated risks just to determine the sex for the parents curiosity. The associated sex linked diseases which could be more accurately diagnosed prenatally from amniocentesis were only researched later, a discovery which made Barr and Bartram's study highly significant. Until their discovery parents were given statistics based on their family histories of sex linked diseases which were nothing more than loose probability. With the sex of the baby able to be determined more reliable statistics could be calculated.

Fuchs and Riis

Steel and Breg

In 1966 Steel and Breg worked together studying the analysis of amniotic fluid. They were the first to successfully culture the cells from amniotic fluid and create a karyotype or chromosome mapping of the DNA of the fetus to study its genetic make up. Their study published in the Lancet in 1966, Chromosome analysis of human amniotic-fluid cells[4], was a major discovery in the progression of amniocentesis as they proved by using a karyotype of the fetus's DNA chromosomal abnormalities were able to be prenatally diagnosed.

Dr. Carlo Valenti

Procedure

Who is eligible for the test?

Since the procedure is invasive and not without a number of risks, the mother is tested before to determine whether she is at a higher risk than average of having a child with a chromosomal or neural tube defect. These tests include:

  • Checking for abnormal ultrasound features.
  • Maternal age is considered, women older than 35 are at a higher risk of having children with chromosomal disorders.[5]
  • History of chromosomal disorders in the family, testing if the parents are Rh positive or Rh negative.[6]
  • History of previous children born with a genetic defect.

When can the test be taken?

An amniocentesis test is most commonly performed between weeks 15 to 16 of gestation which is during the second trimester of pregnancy, however it is able to be performed anytime between weeks 14 to 20. Amniocentesis during the third trimester is strongly avoided as there is a high risk of inducing an early labor from premature rupture of membranes within the womb.[7]. Second trimester procedures are preferred over an early amniocentesis which is performed in the first trimester during weeks 11 to 14 as the earlier the test is performed the higher the risk of miscarriage, as much as 3 times greater. The reason an early amniocentesis option is offered to women is since the delay in tissue analysis of about 2 weeks means if an abortion is required it is physically and emotionally easier earlier on in the pregnancy. If an early diagnostic test is requested Chorionic Villus Sampling (CVS) is suggested as it carries less risk of miscarriage and complications than amniocentesis in the first trimester.[8]


Steps of the procedure

The amniocentesis procedure showing insertion of the needle into the uterus and use of ultrasound.

Preparation

  • Counseling for the mother is provided to investigate any family history of genetic disorders or any birth defects in previous children. The mother is given an assessment[9] on whether this procedure will benefit her based on her history and likelihood of baring a child with a fetal abnormality. She is also informed of the details and risks of the procedure and is able to make an informed decision to continue or not based on anxiety towards an invasive and risky procedure. [10][11]
  • The next step is cleaning of the area before needle insertion with an iodine solution to ensure no contamination.
  • Local anesthetic may be administered but since there is little pain it is usually omitted to avoid a second needle insertion.

Needle Insertion

  • The needle is inserted and 15 mL of amniotic fluid is sampled, it takes approximately 30 seconds to withdraw the fluid.
  • Before the needle is inserted and whilst the needle is inside the uterus ultrasound is used continuously to ensure no harm is caused to the baby or placenta.
  • After the fluid is sampled the baby's heart beat is checked to ensure there was no harm.

The Amniotic Fluid

The process of amniotic fluid analysis in the lab including centrifugation, culturing and karyotyping.
  • The fluid removed contains fetal cells shed from the skin, and the lining of the gastrointestinal and respiratory tracts.
  • These cells which will contain the DNA of the fetus is sent to a lab where it is cultured and the chromosomes are able to be mapped to investigate any chromosomal defects. Protein levels are also analyzed to investigate any neural tube defects.[12]

In the Lab

  • In the lab the amniotic fluid sample is centrifuged to separate the fetal cells from the amniotic fluid containing lipids, proteins, glucose and electrolytes.
  • After it is centrifuged the bottom half of the separated fluid containing the fetal cells is immersed in a culture medium designed to maximize colony growth of the embryonic cells. The cells are able to grow and then stored in an incubator until the time comes to harvest them.[13]
  • The harvested cells are arrested in the metaphase stage of cell division which is the time of most condensation of the DNA. This means the chromosomes are able to be seen under a microscope after correctly dyed.
  • The chromosomes are stained with certain chemicals that bind to the DNA to create a characteristic and recognizable banding pattern that will be different for the varying molecular structures of the chromosomes.

Creating a Karyotype

A karyotype of a human female showing the standard layout of chromosomes, and the abnormality of trisomy of chromosome 21 resulting in Down syndrome.


  • The standard way of observing the stained chromosomes is by creating a karyotype.[13] A karyotype is a photograph taken of the chromosomes arranged from largest to smallest non-sex chromosomes (autosomal) numbered 1 to 22, with the 2 sex chromosomes placed at the end. This allows analysis to be done efficiently and any structural abnormalities to be clearly identified.
  • Structural abnormalities that may be indicative of a genetic disorder include chromosome deletions, duplications, translocations and inversions.



Risks

There are risks involved in the procedure of amniocentesis. There is a chance, however small, of a miscarriage. As stated before, only certain people should undergo the procedure because of these reasons.

A study undertaken dealt with specifically the performance of the amniocentesis procedure over 4 years. It indicated that the number of pregnancies ending in miscarriage due to amniocentesis is as low as 1 in 1000.[14] Also, the number of stillbirths at 32 and 35 weeks is 3%.[15]

This sample space will differ slightly against the actual percentages of the population. What can be observed is that the leading reasons are to find the chromosomal abnormality indicating Down Syndrome and at the request of the mother, possibly due to known family history.

These guidelines allow for properly measuring the risk of the procedure against knowledge that can be gained from it. A doctor or genetic counsellor should be consulted prior to the test.

Timing

Amniocentesis should be performed at a time when enough amount amniotic fluid can be taken for diagnosis without affecting the course of the pregnancy. Generally, this is around weeks 15 -19. As indicated under the procedure section. .[16]


Sometimes there might not be enough amitotic fluid available to take for analysis. In this instance, more amniotic fluid may be taken at a later date.

Infection

There is a risk of infection with amniocentesis. It is intrusive as it requires contact to be made with the amniotic fluid by a needle through the skin, abnormal wall and uterine wall. The transfer of bacteria can occur if the needle and area of needle insertion into the lower abdomen of the woman are not properly sterile.

This risk is lowered by all persons involved in the procedure properly washing their hands and working in a sterile environment. Swabbing the area where the needle is inserted is the most common method of keeping these areas clean .[17]

This also leads to the possibility of transfer of blood plasma from the foetus to the mother. This can cause major issues in the instance where the rhesus (Rh) factor of the baby is positive and the mother is negative. The mother may develop antibodies to Rh factor due to blood transfusion or from a previous birth. The mother's antibodies can diffuse through the placenta and cause red blood cell agglutination. This has devastating effects on the development of the foetus. [18]

It is acceptable to use a needle for pain management of the mother during the procedure. The use of two needles generally increases the risk of infection, so it is more commonly practiced that the mother puts up with the discomfort.

Amniocentesis in first trimester pregnant sheep. The 22 Gauge needle is being guided using a 3.5MHz ultrasound transducer

Use of Ultrasound

Ultrasound is used to show the foetus growing inside the uterus of the mother. The chance of the needle hitting the developing foetus is a risk factor. This can be avoided by constantly using ultrasound to see the position of the developing foetus. The constant vision allows for withdrawing fluid without physically hitting the foetus. It also gives the doctor confidence and reassurance that the needle is in the right place and not causing risk problems.

It also allows the doctor to see that the needle has entered the right area. This reduces the occurrence of piercing other organs such as the bowel, which could lead to infection of the uterus, internal bleeding and other complications which could lead to miscarriage.

Psychological Risks

The emotional state of the mother can have affects on the outcome of the pregnancy. During pregnancy, hormone levels in the mother change to maintain the pregnancy, suppressing the degeneration of the endometrial lining which leads to menstruation.

The invasive nature of amniocentesis can invoke conditions such as anxiety in the mother. This can also be brought on by the results of amniocentesis coming back with undesirable results, leading to anxiety and depression.

Cortisol is a hormone produced in the adrenal gland that is triggered by emotional stressors. If it is produced excessively in the mother, some of the hormones can reach the foetus in the uterus. If this occurs there might be developmental effects on the foetus. These include:

• Asymmetry of coordination of fingers, elbows and others

• Mental development

• Imperfections in nervous system

• Decreased perception, thinking and memory abilities

The presence of a partner during the pregnancy has been found to reduce the stress levels of the mother. However, the elevated anxiety levels brought on by amniocentesis were found not to have the same effect. Therefore health care professionals must monitor the mother closely for signs of anxiety and help them better cope with the situation to reduce this.

Disorders Detected

There are many disorders that can be found from amniocentesis. These can be either chromosomal abnormalities or neural tube defects. The amniotic fluid sample taken is centrifuged so the cells are separated from the plasma. From these cells, the chromosomes are able to be observed and from this, chromosomal abnormalities can be seen.

Disorder Cause Main Caracteristics Occurence Rate (%x10^-3)
Down Syndrome (trisomy 21) The presence of all or a part an extra 21st chromosome Mental retardation

Abnormal musculoskeletal development Particular set of facial characteristics.

1.11
Edwards Syndrome (trisomy 18) The presence of all or a part an extra 18st chromosome Kidney malformation

Structural heart defects Intestines outside body Mental retardation

0.33
Turner syndrome (monosomy X) This is several different conditions, the most common is that an entire sex chromosome is missing Short stature

Swelling in limbs Webbed necks Low set ears

0.4 of girls
Spina Bifida Incomplete closure of the embryonic neural tube at the chorionic end Baby born with protruding spinal chord

Different levels

.7
Anencephaly Failure of neural tube closure at the cranial end No forebrain

Baby born blind, deaf and unconscious

0.0057
Fragile X Faulty X chromosome resulting in failure to express an essential protein for normal neural development from the FMR1 gen Prominent ears, long face, low muscle tone, atypical social behaviour males: 0.27

females: 0.2

Diagnostic Accuracy

Ethical Issues

Current Research

References

  1. <pubmed>18730250</pubmed>
  2. <pubmed>4255486</pubmed>
  3. <pubmed>20325291</pubmed>
  4. <pubmed>4159775</pubmed>
  5. <pubmed>6220164</pubmed>
  6. <pubmed>15832534</pubmed>
  7. <pubmed>18191913</pubmed>
  8. Prenatal Testing: An Amniocentesis Primer. (2004). http://www.plus-size-pregnancy.org/Prenatal%20Testing/prenataltest-amnios.htm
  9. <pubmed>9084385</pubmed>
  10. Brajenović-Milić B, Babić I, Ristić S, Vraneković J, Brumini G, Kapović M. Womens Health Issues (2008) Pregnant women's attitudes toward amniocentesis before receiving Down syndrome screening results.PMID: 18180167
  11. <pubmed>15284930</pubmed>
  12. North East Valley Division General Practice Victoria, Australia.http://www.nevdgp.org.au/info/melb_us/Amniocentesis_melb.htm
  13. 13.0 13.1 <pubmed>5273905</pubmed>
  14. http://www.imff.ie/patient-services-amniocentesis.shtml
  15. <pubmed>PMC2464303</pubmed>
  16. Prenatal Testing: An Amniocentesis Primer. (2004). http://www.plus-size-pregnancy.org/Prenatal%20Testing/prenataltest-amnios.htm
  17. Prenatal Testing: An Amniocentesis Primer. (2004). http://www.plus-size-pregnancy.org/Prenatal%20Testing/prenataltest-amnios.htm
  18. Principles of Human Physiology(2002) William J. Germann Cindy L. Stanfield

2010 ANAT2341 Group Projects

Project 1 - Ultrasound | Project 2 - Chorionic villus sampling | Project 3 - Amniocentesis | Group Project 4 - Percutaneous Umbilical Cord Blood Sampling | Project 5 - Fetal Fibronectin | Project 6 - Maternal serum alpha-fetoprotein | Group Assessment Criteria

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Cite this page: Hill, M.A. (2024, March 28) Embryology 2010 Group Project 3. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/2010_Group_Project_3

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