AUTHOR AND EDITOR INFORMATION

Section 1 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

INTRODUCTION

Section 2 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

Preimplantation genetic diagnosis (PGD) is a technique used to identify genetic defects in embryos created through in vitro fertilization (IVF) before transferring them into the uterus. Because only unaffected embryos are transferred to the uterus for implantation, PGD provides an alternative to current postconception diagnostic procedures, ie, amniocentesis or chorionic villus sampling, which are frequently followed by pregnancy termination if results are unfavorable. PGD is performed in conjunction with IVF and is offered to fertile and infertile couples.

History

Edwards and Gardner successfully performed the first known embryo biopsy on rabbit embryos in 1968. In humans, PGD was developed in the United Kingdom in the mid 1980s as an alternative to current prenatal diagnoses (Harper, 2001). PGD is presently the only option available for avoiding a high risk of having a child affected with a genetic disease without facing the dilemma of pregnancy termination following unfavorable prenatal diagnosis. In 1989 in London, Handyside and colleagues reported the first unaffected child born following PGD performed for an X-linked disorder.

As of May 2001, more than 3000 PGD clinical cycles have been reported. These cycles were performed at more than 40 centers around the world, and almost 700 children have been born, thus demonstrating the reliability and safety of the procedure. PGD is currently available for most known genetic mutations (International Working Group on Preimplantation Genetics, 2001). Since 2001, many more centers have started offering PGD to their patients, unless it is prohibited by the laws of a particular country.

Definition

PGD combines recent advances in genetics and reproductive medicine. For PGD, the early embryo is examined after IVF for inherited diseases or to determine the sex of the embryo for sex-related genetic disorders. The process starts with a basic IVF. When the embryo is at the 6- to 8-cell stage, 1-2 cells (blastomeres) are removed and sent to the genetic laboratory for diagnosis using either polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) techniques, depending on which disease is being sought. The unaffected embryos are then transferred into the mother's uterus.

In certain situations in which the genetic problem is only with the female, a polar body can be removed from the eggs and tested before fertilization.

INDICATIONS AND CONDITIONS

Section 3 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

Indications for PGD

PGD is recommended when embryos may be affected by a certain genetic condition. Only healthy and normal embryos are transferred into the mother's uterus, thus preventing adverse outcomes such as miscarriages, pregnancy termination (after positive prenatal diagnosis), or birth defects (physical and/or mental). Primary candidates for PGD include the following:

• Couples in whom at least one partner has a family history of inheritable genetic disease, carries such a disease, or is otherwise affected by such a disease
• Women 35 years or older (to test for aneuploidy due to maternal age)
• Women with recurrent pregnancy losses, which could be caused by an abnormal chromosomal set coming from either the male or female partner
• Couples with chromosome translocations, which can cause implantation failure, recurrent pregnancy loss, or mental or physical problems in offspring
• Couples with repeated IVF failure
• Men with infertility requiring intracytoplasmic sperm injection (ICSI)

Conditions diagnosed using PGD

PGD should be offered for 3 major groups of disease, including (1) sex-linked disorders, (2) single gene defects, and (3) chromosomal disorders.

Sex-linked disorders

PGD can be used to determine the sex of an embryo in which the specific genetic defect at a molecular level is unknown, highly variable, or unsuitable for testing on single cells. Sex-linked recessive disorders include hemophilia, fragile X syndrome, most of the neuromuscular dystrophies (currently, >900 neuromuscular dystrophies are known), and hundreds of other diseases. Sex-linked dominant disorders include Rett syndrome, incontinentia pigmenti, pseudohyperparathyroidism, and vitamin D–resistant rickets. X-linked diseases are passed to the child through a mother who is a carrier. They are passed by an abnormal X chromosome and manifest in sons, who do not inherit the normal X chromosome from the father. Affected fathers have sons who are not affected, and their daughters have a 50% risk of being carriers if the mother is healthy.

Single gene defects

PGD is used to identify single gene defects such as cystic fibrosis, Tay-Sachs disease, sickle cell anemia, and Huntington disease. In such diseases, the molecular abnormality is detectable with molecular techniques using PCR amplification of DNA from a single cell. Although progress has been made, some single gene defects have a wide variety of rare mutations (eg, cystic fibrosis has approximately 1000 known mutations). Only 25 of these mutations are currently routinely tested. Because most of these rare mutations are not routinely tested, a parent without any clinical manifestations of cystic fibrosis could be a carrier. This allows the possibility for a parent carrying a rare mutation gene to be tested as negative but still have the ability to pass on the mutant cystic fibrosis gene.

Chromosomal disorders

The last group includes chromosomal disorders in which a variety of chromosomal rearrangements, including translocations, inversions, and deletions, can be detected using FISH. Some parents may have never achieved a viable pregnancy without using PGD because previous conceptions resulted in chromosomally unbalanced embryos and were spontaneously miscarried.

Aneuploidy and maternal age

The risk of aneuploidy in children increases as women age. The chromosomes in the egg are less likely to divide properly, leading to an extra or missing chromosome in the embryo (see Table 1). The rate of aneuploidy in embryos is greater than 20% in mothers aged 35-39 years and is nearly 40% in mothers aged 40 years or older. The rate of aneuploidy in children is 0.6-1.4% in mothers aged 35-39 years and is 1.6-10% in mothers older than 40 years. The difference in percentages between affected embryos and live births is due to the fact that an embryo with aneuploidy is less likely to be carried to term and will most likely be miscarried, some even before pregnancy is suspected or confirmed. Therefore, using PGD to determine the chromosomal makeup of embryos increases the chance of a healthy pregnancy and reduces the number of pregnancy losses and affected offspring.

One of the most frequent aneuploidies, trisomy (ie, 3 identical chromosomes present in the embryo), is trisomy of chromosome 21, which leads to Down syndrome. This particular abnormality also frequently leads to spontaneous miscarriages, the precise frequency of which is difficult to determine. Thus, the only reliable information is on the frequency of babies born with Down syndrome. An excellent and informative article in the Journal of the American Medical Association (Hook, 1983) includes information on estimating the incidence of trisomy 21/Down syndrome in fetuses at 16 weeks of pregnancy (also see Table 2).

Table 1. Chromosomal Abnormalities

Table 2. Frequency of Down Syndrome Per Maternal Age

PGD and sex selection unrelated to disease

Because PGD can help determine the sex of the embryo, many couples request PGD for sex selection, which can be motivated by cultural, social, ethnic, psychological, and other reasons such as the simple desire to have children of a preferred sex to achieve a balance among children in a given family or to determine a sex birth order.

The use of PGD for sex selection unrelated to disease is controversial and has elicited moral outrage about not implanting normal embryos when they are found to be of an undesired sex. Some people consider this infanticide. Other frequent objections include the danger of sex discrimination, the perpetuation of oppression against females, the ethics of expanding control over nonessential characteristics of offspring, and the relative importance of sex selection when weighed against medical and financial burdens to parents. Much discussion is still necessary to achieve a reasonable consensus and acceptance of PGD for sex selection. Personal, religious, ethical, and moral norms vary among different populations. Personal preferences must be considered and proper respect given to them.

PROCESS

Section 4 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

PGD process using blastomere

Workup

Before requesting PGD, candidates should consult a geneticist or genetic counselor to evaluate the risk of transferring their genetic abnormality to their offspring. Tests should be performed to confirm the diagnosis of the affected parent, to pinpoint the genetic change leading to the condition in question, and to ensure that the currently available technology can identify that genetic change in a polar body or a blastomere biopsy sample from a 6- to 8-cell embryo/blastocyst (see Image 1).

In vitro fertilization

A 16- to 17-gauge needle is typically used for transvaginal aspiration of the follicles under ultrasound control. Vaginal tissue sensitivity is low, whereas ovarian sensitivity is considerably higher. The most sensitive part of the pelvis is the peritoneal lining. Light general anesthesia or conscious sedation with a narcotic and tranquilizer are used, depending on physician and patient preferences. Both are satisfactory for egg retrieval. Patients are kept supine until they are fully awake and all vital signs are stable. They should be discharged to the custody of an adult and are strongly advised to not engage in certain activities, such as driving, for the remainder of the day.

The IVF procedure consists of the following steps:

1. Ovarian stimulation is needed in order to produce several good-quality eggs. During the 7- to 14-day stimulation period, ultrasound examinations and laboratory tests are frequently performed to monitor the development and maturation of the follicles.
2. When the follicles are ready for egg retrieval, the patient is given a pain medication. Under ultrasonographic or laparoscopic guidance, the follicles are punctured, their contents aspirated, and the eggs identified and harvested. The procedure usually lasts less than 30 minutes.
3. The male partner typically provides a semen sample by masturbation.
4. The eggs are cultured for a few hours after retrieval to allow for final maturation to occur. A polar body can then be removed. The sperm is subsequently washed to allow the capacitation procedure. After either swim-up or radiant purification of the sperm sample, a small aliquot containing approximately 50,000 sperm/mL is added to the egg-containing dish. For the PGD procedure, ICSI is preferred.
5. The following morning, the eggs are examined for signs of fertilization, which usually include the formation of 2 pronuclei. These nuclei represent the male and female contribution to the embryo.
6. On day 3, when the embryo is normally at the 8-cell stage, the embryos are prepared for biopsy. Normal development includes progress to the 2-cell stage, the 4-cell stage, and, by the third day, usually 6-10 cells, exhibiting homogenous cytoplasm (see Image 2).

Removal of the single cell

Most clinics offering PGD perform biopsy using one of two techniques:

• Polar body biopsy
• • Polar body biopsy works only for female chromosomal disorders. The adult egg produces 2 small cells called polar bodies. One of these cells can be removed and tested, providing information on the chromosomal content of the egg.
  • Only information about the mother can be obtained by analyzing polar bodies; however, chromosomal abnormalities occurring after fertilization (when the sperm meets the egg) are not detected.
• Blastocyst biopsy
• • Blastocyst biopsy is accomplished using the micromanipulating microscope, which allows extraction of a single blastomere from a developing embryo.
  • Before extracting the single cell from an 8-cell embryo, the embryo is incubated in calcium- and magnesium-free medium for approximately 20 minutes in order to reduce blastomere-to-blastomere adherence.
  • The embryo is then anchored on one side with a holding pipette; simultaneously, a manipulation pipette containing acidic Tyrode solution is placed adjacent to the zona pellucida. The Tyrode solution is gently extruded so that the zona can be thinned and a small opening can eventually be made. The embryation pipette is placed through this opening and focused on the blastomere of choice, which should contain a visible nucleus. The blastomere is subsequently gently aspirated into the pipette and expelled into the medium.
  • The embryo, now containing 7 cells instead of 8, is returned to the incubator into the appropriate culture medium. The blastomere is then processed for either FISH or PCR, depending on the genetic condition to be studied.
  • • For FISH, the cell is placed into a hypertonic solution containing sodium citrate native bovine serum albumin to allow for swelling of the cell. The cell is then transferred to a slide, which allows the cell to spread as it dries. A fixative of methanol acidic acid is applied to it to make the cellular contents disappear or wash away, leaving only the chromosomal component.
    • For PCR, the cell is placed into a special small PCR tube containing PCR buffer, which allows a reaction for replication and amplification of the genetic signal.
  • The removal of the blastomere is a technically challenging procedure. The embryologist's goal, accomplished using a special microscope and micromanipulators, is to remove an intact cell with minimal trauma to the remaining embryo (see Image 3).

Genetic testing

Polymerase chain reaction

PCR is used for the diagnosis of single gene defects, including dominant and recessive disorders. PCR, sometimes called DNA amplification, is a technique in which a particular DNA sequence is copied many times in order to facilitate its analysis. PCR rapidly multiplies a single DNA molecule into billions of molecules.

The DNA is immersed in a solution containing the DNA polymerase enzyme, unattached nucleotide bases, and primers. The solution is heated to break the bonds between the strands of the DNA. When the solution cools, the primers bind to the separated strands, and the DNA polymerase quickly builds new strands by joining the free nucleotide bases to the primers. By repeating this process, a strand that was formed with one primer binds to the other primer, resulting in a new strand that is specific solely to the desired segment. Further repetitions of the process can produce billions of copies of a small piece of DNA in several hours.

PCR is a relatively fast and convenient way to test DNA. The method has been used in a variety of preimplantation genetic testing protocols. However, it requires sufficient amounts of a pure, high-quality sample of DNA, which is sometimes difficult to obtain from a single cell such as a polar body or blastomere. In addition, laboratory contamination and allele dropout are possible complications.

Only one cell should be amplified; however, if another cell or piece of DNA enters the tube, it is also amplified. ICSI must be used to minimize this problem and to ensure that no excess sperm are present (paternal contamination) and that all the cumulus cells have been removed (maternal contamination).

The laboratory environment must be strictly controlled to avoid the introduction of contaminants to the tested material. The laboratory technicians must be trained extremely well to avoid all types of outside interferences.

Allele dropout is the preferential amplification of one allele over another and is mainly a problem for PGD of dominant disorders or when 2 different mutations are carried for a recessive disorder and only one mutation is being analyzed. Each mutation or disease requires a specific PCR test to be developed, which is a time-consuming and expensive process.

Fluorescence in situ hybridization

FISH is used for the determination of sex for X-linked diseases, chromosomal abnormalities, and for aneuploidy screening. Probes, ie, small pieces of DNA that are a match for the chromosomes being analyzed, bind to a particular chromosome. Each probe is labeled with a different fluorescent dye. These fluorescent probes are applied to the cell biopsy sample and are expected to attach to the specific chromosomes. They can be visualized under a fluorescent microscope. The number of chromosomes of each type (color) present in that cell is counted. The geneticist can thus distinguish normal cells from abnormal cells, such as those with aneuploidy (see Images 4-5).

A summary of PGD applications categorized by PCR or FISH is as follows:

• Polymerase chain reaction
• • Single gene defects in autosomal disease
  • Single gene defects in male infertility
  • Identification of sex in X-linked diseases (American Society for Reproductive Medicine, 2001)
• Fluorescence in situ hybridization (preferred because PCR bears the risk of misdiagnosis caused by contamination)
• • Aneuploidy screening in women of advanced maternal age
  • Aneuploidy screening for male infertility
  • Identification of sex in X-linked diseases
  • Recurrent miscarriages caused by parental translocations

Embryo transfer

After the genetic laboratory provides detailed information, the future parents, along with their physicians, decide which embryos should be transferred or frozen and which should not be used. Usually, 2-4 embryos are transferred, depending on the couple's specific conditions.

The procedure takes just a few minutes and does not require anesthesia or analgesia. The physician inserts a speculum into the vagina and cleans the cervix, removing all cervical mucus. In some situations, washing with an appropriate fluid is required to facilitate the cleaning of the endocervical canal. A thin plastic catheter, into which the embryologist has transferred the embryos, is then advanced near the top of the uterus. Recently, ultrasound has been used to achieve more precise placement of the embryos. Once the placement of the catheter is correct, preferably 1-2 cm from the top of the uterine cavity, the embryos are expelled from the catheter and deposited into the uterus.

After transfer of the embryos, patients usually rest in mild Trendelenburg position on the transfer table for 1-3 hours. The patient is advised to rest during the first 24 hours after the embryo transfer and to engage in only limited activity during the second 24 hours. She may then return to her normal activity level. Note that physical activity or diet has only limited impact on embryo implantation or conception. Once the embryos are transferred, a patient can do little to influence the outcome of her cycle.

The patient needs to be followed carefully to ensure that hormonal levels, which are essential in sustaining the pregnancy, are appropriate. The concentration of the hormones in blood is tested on frequent occasions, and when necessary, hormonal levels are maintained or improved by exogenous hormones, most likely progesterone. Because of the complicated metabolism of progesterone, a vaginal or intramuscular application is preferred. When necessary, the production of essential hormones by the ovaries might be improved by the administration of human chorionic gonadotropin in the luteal phase of the cycle. Many pregnancies can be "rescued" by carefully monitoring the patient after embryo transfer.

CONSIDERATIONS AND CONTROVERSIES

Section 5 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

Considerations and challenges

• Fertile patients must undergo IVF to produce suitable embryos.
• Success depends on a number of good embryos.
• Biopsy can be problematic.
• Even with a successful IVF and PGD procedure, pregnancy is not guaranteed after transfer, nor is a term or near-term delivery.
• Removal of a single cell without breaking it or causing serious damage is technically difficult and requires skill and experience.
• The diagnostic methodology for a new disease is a time-consuming and expensive process.
• Analysis of a single cell has limitations, and misdiagnosis resulting from mosaicism (when the embryo has cells with different compositions) may occur. For this reason, prenatal diagnosis may be considered to confirm the condition of the fetus.
• A relatively large number of eggs or embryos may be found to be abnormal, thus leaving only a few or no healthy embryos for transfer.
• Not all chromosomal or genetic abnormalities can be diagnosed with PGD because only a restricted number of chromosomes can be examined at one time during the course of a single procedure.
• Damage to the embryo (projected to be 0.1%) may accidentally occur during removal of the cell.
• Currently, only a specific examination of a single biopsied cell is available. A single cell cannot be screened for multiple genetic conditions.

Controversies

• Some people fear that the examination of an embryo may be misused for discrimination. Some worry that PGD can be used to design the "perfect baby."
• Some people are concerned that the single removed cell could have developed into a fetus but was destroyed by the test. Some consider this murder.
• Fertilized cells found to carry a genetic defect are not implanted and are allowed to die in the dish.
• Some genetic diseases manifest when the host is aged 30-40 years or even older. Critics argue that a cure might be found during the interim period.
• Some people consider the use of PGD for sex selection for social and personal reasons to be inappropriate interference with the natural selection of progeny.

ADVANTAGES

Section 6 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

Currently available technology can help eliminate some genetic diseases in the future (eg, Tay-Sachs disease, cystic fibrosis, Huntington disease, X-linked dystrophies). Complete cures for many genetic diseases are not likely to be found soon; therefore, preventing the disease is preferable to waiting for a possible cure to eventually become available. Furthermore, available treatments often have multiple adverse effects. Prolonging the lifespan of affected patients could cause them to develop diseases not previously known to be associated with the particular genetic condition (eg, diabetes, osteoporosis). For instance, as improved treatment prolongs life for individuals with cystic fibrosis, other manifestations of the pancreatic insufficiency and nutritional malabsorption associated with the disease, such as diabetes and osteoporosis, begin to emerge.

Most testing for genetic diseases is currently performed through amniocentesis or chorionic villus testing when the fetus is aged 10-16 weeks. If the examination findings reveal a genetically defective fetus, the options available to parents are to have a child with a genetic disease or to have an abortion. This is a difficult and often traumatic decision, especially in advanced pregnancy. However, PGD is performed before pregnancy begins, thus eliminating this dilemma.

Persons with a genetic disease or those who know that they are carriers frequently choose to not have children in order to avoid the risk of passing on the disease. PGD allows them the opportunity to have a child free of their particular disease.

When PGD becomes more widespread and is accepted and recommended by support societies, the number of babies born with diagnosable genetic diseases should fall dramatically. This will represent a significant reduction of physical and emotional stress related to care for an affected family member and a significant reduction of medical expenses to society. For example, the cost of lifetime medical care of a person with cystic fibrosis is more than $2 million.

The expansion of genetic investigation will also open new avenues of understanding of various previously unknown conditions and associations. In 2001, the US National Institutes of Health and the American College of Obstetricians and Gynecologists recommended testing of patients of childbearing age for cystic fibrosis (American College of Obstetricians and Gynecologists, 2001). Voluminous information will become available from these examinations. Hopefully, other genetic diseases will also become intensively studied in the near future.

FUTURE

Section 7 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

The first successful cases of PGD in humans were performed in 1988. However, the development and acceptance of PGD since then has been slow, mainly due to the time necessary to develop and learn single-cell diagnostic techniques and to the costs involved. PGD is a relatively new procedure, and much ongoing research is being performed to expand and improve it. However, much more work also must be completed before PGD becomes a more comprehensive, accepted, and widely available procedure.

Almost weekly reports on identification of genetic changes tied to various diseases are published in scientific and lay literature. In the future, genetic links to common diseases (eg, diabetes, hypertension, cardiovascular diseases, endometriosis, cancers) may be identified, and PGD will become available to control the transmission of these diseases to future generations. This will gradually lead to the complete eradication of many dreadful genetic diseases.

Two new procedures reportedly examine all the chromosomes from a single cell at one time: comparative genomic hybridization and interphase conversion.

Comparative genomic hybridization

In comparative genomic hybridization, the embryo nucleus is labeled with a fluorescent dye and a control cell is labeled using another color, ie, red or green. The 2 are then cohybridized onto a control metaphase spread, and the ratio between the 2 colors is compared. If the chromosomal analysis shows an excess of red, the embryo nucleus contains an extra chromosome. If an excess of green is apparent, then the embryo nucleus is missing one of these chromosomes. Currently, this technique takes 72 hours, and embryo cryopreservation is necessary to provide the time necessary to undertake the diagnosis. A successful case has been reported in Australia.

Interphase conversion

In interphase conversion, an interphase nucleus is converted to metaphase by fusing the interphase nucleus with an enucleated oocyte (eg, bovine). When the interphase nucleus is induced into metaphase, all the chromosomes in the nucleus can be examined.

For PCR, fluorescence multiplex PCR has already been applied to PGD cases. DNA chip technology allows more genes to be analyzed. However, the value of this new technology for PGD is limited because the mutation considered to be present is known before PGD is performed and only a few polymorphic markers are required. However, increasing the number of diseases that can be diagnosed by single-cell PCR is crucial so that more patients can benefit from PGD.

MULTIMEDIA

Section 8 of 9  

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

Media file 1:  Workup.
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Media file 2:  Aspiration, fertilization, and transfer.
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Media file 3:  Removal of blastomere from an 8-cell embryo (blastocyst).
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Media file 4:  Abnormalities of chromosomes 13, 18, and 21.
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Media file 5:  Multiple fluorescence in situ hybridization demonstrating chromosomes 13 (triploid), 18, 21, and Y.
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Media file 6:  Fluorescence in situ hybridization (FISH) analyses.
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Media file 7:  Autosomal dominant inheritance.
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Media file 8:  Autosomal recessive inheritance.
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Media file 9:  X-linked dominant inheritance.
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Media file 10:  X-linked recessive inheritance.
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Media file 11:  Polymerase chain reaction with regard to reproduction and genetics.
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Media file 12:  Fluorescence in situ hybridization with regard to reproduction and genetics.
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Media file 13:  Reasons and timing for gender selection.
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Media file 14:  Possibilities for cure, treatment, and prevention through preimplantation genetic diagnosis.
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Media file 15:  Instruments used for preimplantation genetic diagnosis compared with a human hair (upper part of picture). To the lower-left side is the holding pipette, and to the right side is the glass needle used for aspiration of the blastomere.
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Media file 16:  Intracytoplasmic sperm injection. Puncture of the oocyte.
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Media file 17:  Intracytoplasmic sperm injection. Injection of sperm.
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REFERENCES

Section 9 of 9

• Authors and Editors
• Introduction
• Indications and Conditions
• Process
• Considerations and Controversies
• Advantages
• Future
• Multimedia
• References

• American College of Obstetricians and Gynecologists, American College of Medical Genetics. Preconception and Prenatal Carrier Screening for Cystic Fibrosis: Clinical and Laboratory Guidelines. Washington, DC; American College of Obstetrics and Gynecology;. October, 2001.
• American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. A practice committee report: Preimplantation genetic diagnosis. Birmingham, Ala: American Society for Reproductive Medicine;. June, 2001.
• Baird DD, Weinberg CR, McConnaughey DR, Wilcox AJ. Rescue of the corpus luteum in human pregnancy. Biol Reprod. Feb 2003;68(2):448-56. [Medline].
• Edwards RG, Gardner RL. Choosing Sex Before Birth. New Scientist. 1968;38:218-20.
• Ethics Committee, American Society of Reproductive Medicine. Sex selection and preimplantation genetic diagnosis. The Ethics Committee of the American Society of Reproductive Medicine. Fertil Steril. Oct 1999;72(4):595-8. [Medline].
• Flinter FA. Preimplantation genetic diagnosis. BMJ. Apr 28 2001;322(7293):1008-9. [Medline].
• Handyside AH, Pattinson JK, Penketh RJ, et al. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet. Feb 18 1989;1(8634):347-9. [Medline].
• Harper JC. Introduction. In: Harper JC, Delhanty JDA, Handyside AH, eds. Preimplantation Genetic Diagnosis. London, UK: John Wiley & Sons; 2001:. 3-12.
• Hook EB, Cross PK, Schreinemachers DM. Chromosomal abnormality rates at amniocentesis and in live-born infants. JAMA. Apr 15 1983;249(15):2034-8. [Medline].
• International Working Group on Preimplantation Genetics, International Congress of Human Genetics. Preimplantation Genetic Diagnosis: Experience of Three Thousand Cycles. Report of the 11th Annual Meeting of International Working Group on Preimplantation Genetics, in association with 10th International Congress of Human Genetics. Vienna, Austria; May, 2001. [Full Text].
• Kahraman S, Karlikaya G, Sertyel S, et al. Clinical aspects of preimplantation genetic diagnosis for single gene disorders combined with HLA typing. Reprod Biomed Online. Nov 2004;9(5):529-32. [Medline].
• Munne S, Cohen J, Sable D. Preimplantation genetic diagnosis for advanced maternal age and other indications. Fertil Steril. Aug 2002;78(2):234-6. [Medline].

Article Last Updated: Apr 13, 2005