4
Assisted Reproductive Technology

In this chapter, we address the following question in our task statement:

To what extent can our knowledge of assisted reproductive technologies inform the debate on human cloning?

To organize its response to that question, the panel developed a series of subquestions, which appear as the section headings in the following text.

WHAT IS ASSISTED REPRODUCTIVE TECHNOLOGY?

Assisted reproductive technology (ART) refers to any treatment or procedure for assisting reproduction that includes the handling of human eggs, sperm or embryos, such as in vitro fertilization (IVF).

HOW EFFICIENT IS IN VITRO FERTILIZATION? HOW DOES IT COMPARE IN EFFICIENCY WITH ANIMAL CLONING?

IVF involves the mixing of egg and sperm in the laboratory to generate embryos suitable for transfer to a uterus 2 or 3 days later. An IVF cycle in humans usually involves the transfer of at least two embryos at a time. In the United States in 1998, 20% of human IVF transfers involved one or two embryos, 33% involved three embryos, 28% involved four embryos, and 19% involved five or more embryos [1].

Of all the reported IVF cycles in the United States in 1998 using fresh eggs and embryos derived from the patient, 30.5% resulted in pregnan-



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Scientific and Medical of Aspects: Human Reproductive Cloning 4 Assisted Reproductive Technology In this chapter, we address the following question in our task statement: To what extent can our knowledge of assisted reproductive technologies inform the debate on human cloning? To organize its response to that question, the panel developed a series of subquestions, which appear as the section headings in the following text. WHAT IS ASSISTED REPRODUCTIVE TECHNOLOGY? Assisted reproductive technology (ART) refers to any treatment or procedure for assisting reproduction that includes the handling of human eggs, sperm or embryos, such as in vitro fertilization (IVF). HOW EFFICIENT IS IN VITRO FERTILIZATION? HOW DOES IT COMPARE IN EFFICIENCY WITH ANIMAL CLONING? IVF involves the mixing of egg and sperm in the laboratory to generate embryos suitable for transfer to a uterus 2 or 3 days later. An IVF cycle in humans usually involves the transfer of at least two embryos at a time. In the United States in 1998, 20% of human IVF transfers involved one or two embryos, 33% involved three embryos, 28% involved four embryos, and 19% involved five or more embryos [1]. Of all the reported IVF cycles in the United States in 1998 using fresh eggs and embryos derived from the patient, 30.5% resulted in pregnan-

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Scientific and Medical of Aspects: Human Reproductive Cloning cies, and 82% of these pregnancies (25% of all cycles) resulted in live births [1]. Although efficiencies are not usually reported as the fraction of successful pregnancies per embryo transferred, 12% of embryos transferred in one study after preimplantation genetic diagnosis (PGD) implanted successfully (yielding a success rate of 19.9% when measured in the usual terms of pregnancy per cycle) [2]. Clinical characteristics of the male and female partners play a major role in determining the success rate of IVF treatment. For example, in 1994, the highest success was reported for couples in which the female partner was younger than 40 years old and the male had a normal semen analysis (24.5% live births per cycle). The lowest success was reported for women older than 40 years old with a male partner with a normal semen analysis (9% live births per cycle) or abnormal semen analysis (8.5% live births per cycle) [3]. The success rate of IVF may be constrained by the relatively high rate of pregnancy loss in humans. In unassisted reproduction, many pregnancies are lost before there is any clinical sign of their existence (“occult pregnancies”), and additional pregnancies are lost after they are detectable with hormone measurements but before they are detectable with ultrasonography (“chemical pregnancies”). According to one source [4], “more than 80% of [spontaneous] abortions occur in the first twelve weeks, and the rate decreases rapidly thereafter.” This contrasts with the frequent loss of cloned animal fetuses late in gestation. IVF procedures involve the collection of eggs for fertilization. Any human reproductive cloning attempt would also involve this procedure, and the low efficiency of animal cloning suggests that a large number of eggs would have to be collected. The collection of these eggs would bring with it the risk of ovarian hyperstimulation syndrome in donors. The incidence of moderate and severe cases of this syndrome in studies in which more than 1000 IVF cycles were evaluated ranges from 0.8% [5] to 1.95% [6]. Maternal death resulting from the syndrome is rare enough that it is the subject of occasional case reports. In the United States, multiple embryos are frequently implanted during an IVF cycle to increase the chances of a successful pregnancy [1]. That often results in multiple births, which are associated with risks of morbidity and mortality for the mother and, because of prematurity and low birth weight, for the children. When IVF was first adopted in humans, no increase in the frequency of major malformations had been seen in IVF experiments in mice relative to normal animal reproduction [7]. That situation is in contrast with the data on animal cloning discussed in Chapter 3; cloned animals have markedly more problems, particularly severe abnormalities throughout gestation, than those animals produced by normal reproduction.

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Scientific and Medical of Aspects: Human Reproductive Cloning WHAT OTHER ART PROCEDURES ARE RELEVANT TO HUMAN REPRODUCTIVE CLONING? WHAT IS THEIR RELEVANCE? Blastocyst culture and transfer involve the growth of preimplantation embryos for 5 or 6 days before transfer to a uterus [8]. People who wish to clone humans might take advantage of this technique for two reasons: to try to extend the time available for carrying out preimplantation genetic diagnosis without freezing the embryos and to improve implantation rates. Intracytoplasmic sperm injection (ICSI) is a method in which a single sperm or sperm-precursor cell is injected directly into an unfertilized egg. It is used in cases of severe male factor infertility. The possibility has been raised that sperm will not set up or maintain all necessary male imprints before being injected in ICSI [9; 10]; this is a concern particularly if the sperm are isolated at an early stage of development (from testes rather than ejaculate) [11]. There have been reports of more frequent congenital defects [12] and delayed mental development [13] in some children conceived through ICSI, although both reports have been contested [14; 15]. Other clinicians, after controlling for the effects of multiple births and parental age, have observed no increased risks after ICSI relative to other ART procedures when they scored for congenital malformations [16] (except an increased risk of a genital malformation termed hypospadias possibly related to paternal subfertility [16]) [16-18], obstetric outcome [19; 20] or neurodevelopment [21]. Furthermore, a small study of one particular DNA location did not reveal any imprinting defects after ICSI [22]. Additional research is needed, however, to assess imprinting at multiple genomic sites and to determine the relevance to pregnancy outcome of imprinting status at these sites. If ICSI does lead to imprinting problems, it would suggest that human eggs are incapable of ensuring that the correct pattern of sperm-derived imprints are established or maintained. Similar failures in imprinting after cloning could result in birth defects. ICSI does cause a minor increase in the frequency of sex-chromosome abnormalities [23; 24], but this is probably a result primarily of genetic defects inherited from the infertile father [25-28] and unrelated to concerns about imprinting. Ooplasmic transfer involves the transfer of a small amount of cytoplasm from a fresh donor egg (one that has never been frozen) into a recipient egg that for some reason (such as age or mitochondrial abnormalities) is defective for fertilization or postfertilization development. The success of this technique in producing a live human birth [29; 30] suggests that the mixing of cytoplasm from two different cells, as occurs in reproductive cloning, does not necessarily cause problems. It is important to note, however, that the donor cytoplasm in ooplasmic transfer comes

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Scientific and Medical of Aspects: Human Reproductive Cloning from another egg, whereas the cytoplasm that might come along with the donor nucleus in nuclear transplantation is derived from a somatic cell. Oocyte nuclear transplantation involves the transfer of an egg nucleus into a fresh egg that lacks its own nucleus. It differs from cloning in that the nucleus is derived from a normal egg rather than a diploid somatic cell, and the procedure is followed by fertilization by a normal haploid sperm. If oocyte nuclear transplantation were successful, however, it would suggest that a nuclear transplantation step itself, and the associated manipulations—such as embryo culture, nuclear extraction, and nuclear injection—do not preclude the birth of healthy babies. Oocyte nuclear transplantation has resulted in live births in mice, although the mice have shown growth deficiencies [31]. The procedure has also been carried out in humans, but the resulting blastocyst was terminated [32], and further experimentation was prohibited by the Food and Drug Administration (FDA) [33]. Embryo assessment is the process by which embryos are graded visually for their rate of cell division and degree of “intactness” and therefore likelihood of successful implantation [34; 35]. Those who wish to attempt reproductive cloning might want to take advantage of similar techniques to reduce the number of failed transfers. However, it is not possible to predict which of the embryos deemed intact by embryo assessment will implant successfully [36], so this method will be of limited use to those attempting human reproductive cloning, as is the case for IVF. People who wish to clone humans with any of those approaches might want to implant multiple embryos, as is frequently done in IVF, to increase the chances of a successful pregnancy. As in IVF, the resulting increase in multiple births would be expected to cause considerable risks of morbidity and death for the child (because of prematurity and low birth weight) and the mother. The risk to the mother might be increased by the possibility of multiple overweight fetuses. CAN CURRENT ART PROCEDURES BE USED TO ASSESS POSSIBLE RISKS ASSOCIATED WITH CLONING? No current ART procedure mimics identically the risks inherent in cloning, because current ART procedures all deal with some form of combining sperm and egg and therefore do not give rise to the widespread problems with reprogramming or imprinting that are expected in cloning [37]. The first successful live human birth after IVF was in 1978 [38]. ART procedures, such as IVF, are still new enough that possible long-term effects (for example, adult disorders among the offspring, or disorders in

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Scientific and Medical of Aspects: Human Reproductive Cloning the children born to IVF children) remain unknown. Studies have not turned up major problems if such factors as the mother’s age and the occurrence of multiple pregnancies are taken into account [39], except for an approximately three-fold increase in the frequencies of three very rare conditions (neural tube defects, alimentary atresia and omphalocele) [39]. DOES CLONING PROVIDE BENEFITS NOT PROVIDED BY CURRENT ART PROCEDURES? With current ART procedures, many people are capable of having a child to whom they have at least some genetic link. Exceptions include people who lack any germ cells because of severe infertility. Human reproductive cloning would provide an alternative for these people. Future options for those who lack any germ cells may include the use of artificial gametes, where a diploid adult nucleus is reduced to a haploid state before combination with an oocyte haploid genome (although this may result in the same abnormalities seen in animal cloning procedures), and the transfer of male germ cells from donors to testes of sterile men. CAN THE SCREENING METHODS USED IN ART PROCEDURES BE USED TO PREVENT POTENTIAL SEVERE DEFECTS IN REPRODUCTIVELY CLONED HUMANS? Preimplantation genetic diagnosis Preimplantation genetic diagnosis is performed 2-4 days after fertilization on one or two cells removed from the developing preimplantation embryo [40-45]. Whole-genome amplification [46] can be used as an initial step to increase the amount of DNA available for analysis. Chromosomal abnormalities and specific, preidentified mutations can be detected before implantation of a normal embryo. At least in a research context [46], it is possible to start from a single cell’s worth of DNA and get sufficient amplification to allow for accurate quantification with comparative genomic hybridization. Researchers have projected that this technique can be abbreviated to make it compatible with the limited time available for preimplantation genetic diagnosis [43], and the same could be true for related techniques that use RNA as a starting material. However, technical challenges must be overcome and accuracy and utility demonstrated. A similar analysis could be performed on reproductively cloned embryos, but the emphasis would be on detecting errors caused by defective reprogramming or imprinting. (It is important to recognize that any genetic defect present in the nucleus donor, such as a mutation in a gene required for fertility, would be reproduced in the cloned offspring.) Tests

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Scientific and Medical of Aspects: Human Reproductive Cloning for defective reprogramming or imprinting have not been reported in connection with current preimplantation genetic diagnosis, so the appropriate method would have to be developed first. (At the meeting on August 7, the panel was told that such methods had been developed and applied, but no details were provided [47].) Furthermore, the probable location of the errors would not be known ahead of time. Most genes important for placental function are not active in the morula [48], the only stage when cells can be taken for preimplantation genetic diagnosis, so the functioning of these genes could not be tested with these procedures. For genes that are active in the morula, two tests would be important: Expression levels. The amount of RNA or protein product made by each gene should be tested in screens that are capable of assaying for thousands of genes or proteins. The levels should match those seen in normally fertilized embryos. To allow detection of gene transcripts present in low abundance in the embryo, the RNA molecules would first have to be amplified, but this amplification step could be unequal for different RNAs (because of variation in the efficiency of primer hybridization and other factors) and therefore introduce errors [49-51]. Imprinting levels. This test will be especially difficult in the context of preimplantation genetic diagnosis because the methods used to increase the tiny amounts of DNA available from single embryo cells are currently a challenge for imprinting tests. The location of many imprinted areas in the human genome and the total number of imprinted genes remain unknown [52]. In addition, the observation that imprinting can occur later in development and at dissimilar times in different tissues suggests that examination of imprinting in early embryos might not provide adequate information. Early embryos often have a mixture of cells, of which some have defects and some do not. Thus, if a given cell is found to lack reprogramming and imprinting errors, it does not guarantee that other cells in the embryo will not have problems. Postimplantation screening Screening after implantation is done by acquiring cells through amniocentesis, chorionic villus sampling (CVS), or recovery from maternal blood [53-55]. As with preimplantation genetic diagnosis, cloned embryos would need to be screened for expression levels and imprinting defects. The technical challenges here would be reduced in that more cells would be available for analysis, but they would be complicated because imprinting patterns differ between the embryo and the placenta.

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Scientific and Medical of Aspects: Human Reproductive Cloning Testing of fetal cells would have to be done with a sample from amniocentesis or maternal blood rather than CVS, because CVS samples placental cells. But testing of placental tissue with CVS might also be important. If human embryonic cells develop a problem, they often become incorporated preferentially into the placenta [56]. The presence of such defective cells in the placenta can be an indicator that a rarer subset of cells in the embryo proper is defective. Placental defects might become apparent at many times during gestation, but in current clinical practice CVS is used only during a narrow time period. (CVS is not used earlier, for fear of causing problems with the pregnancy; and it is not used later, because of a desire to induce any necessary abortion as early as possible in the pregnancy.) The errors in reprogramming seen in cloned cattle and mouse embryos [57-59] suggest that few cloned embryos will have a perfect expression profile. It is not clear how the “best” embryos would be selected from such an imperfect pool. Errors in the methylation of genes have been seen in both the placenta and tissues of cloned mice [60; 61]. These errors, which involved only about 0.5% of over 1000 DNA regions screened, varied from mouse to mouse and appeared to be random. However, it is not known whether the errors are associated with specific abnormalities [60; 61]. Modifications of imprinting occur in some specific tissues (such as the brain) later in development [62; 63]. It might be impossible to test for the correct occurrence of these modifications, and others occur too late for abortion to be considered. Some cloned animals have developed additional problems (such as late-onset obesity and immune problems; see also Chapter 3) as they have been observed longer. TO WHAT EXTENT ARE ART PROCEDURES REGULATED IN THE UNITED STATES? Reproductive cloning can be considered an assisted reproductive technique and thus may be subject to any regulations that cover existing ART procedures. In the United States, ART procedures have generally been subject to minimal oversight and regulation [64-66]. The reasons include a lack of federal funding (and thus lack of institutional review board activity), a lack of FDA review, noncoverage of ART procedures by health-insurance companies, and a paucity of medical malpractice litigation because some level of failure is expected in ART procedures. Unlike some countries, the United States does not have a structure for evaluating experimental ARTs as they are developed. Nor is information publicly available on the total number of eggs retrieved, the number of embryos donated for research in IVF clinics, or what studies are per-

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Scientific and Medical of Aspects: Human Reproductive Cloning formed on them. The United Kingdom, in contrast, licenses research and clinical services involving IVF [67] via the Human Fertilisation and Embryo Authority [68]. The Fertility Clinic Success Rate and Certification Act of 1992 provides the only means for national oversight of ART procedures in the United States. That federal legislation requires ART clinics and embryo laboratories to report their pregnancy success rates and follow good laboratory practices [69]. These and other data covering United States ART clinics are published yearly under peer review in Fertility and Sterility [3; 70-75] and form the basis of a database that was established in 1987 by the Society of Assisted Reproductive Technologies (SART), an affiliated society of the American Society for Reproductive Medicine (ASRM). Since 1995, SART has collected the data annually from the 373 IVF programs (of about 400 total programs in the United States) that are SART members. These data are provided to the Centers for Disease Control and Prevention (CDC), which analyzes and publishes them, making them available on its website [1]. HAVE ANY ART PROCEDURES EVER BEEN PROHIBITED OR THREATENED WITH PROHIBITION? In the past, ART procedures have frequently faced opposition and bans that were later lifted. In the 1950s and early 1960s, state bills were introduced to ban, and in some cases criminalize, donor insemination. Similar opposition occurred when IVF was introduced in the 1970s. Both are common procedures today. The concept of surrogate motherhood was introduced in the 1980s, and some state laws ban surrogacy contracts [67]. FINDINGS 4-1. Reproductive cloning efficiencies observed in animals are variable and extremely low compared with efficiencies seen with current human IVF. 4-2. Current techniques for embryo assessment are of limited use in determining the likelihood of successful implantation of a particular embryo. 4-3. No current ART procedure mimics the risks inherent in reproductive cloning, because reproductive cloning involves the use of somatic rather than germ-cell nuclei. 4-4. Tests to detect all the possible errors in imprinting and reprogramming do not exist. Such tests would be difficult to adapt to the small

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Scientific and Medical of Aspects: Human Reproductive Cloning amount of material and the short period available for preimplantation diagnosis. 4-5. ART procedures have been minimally regulated in the United States, and the lack of regulation has resulted in a shortage of data pertaining to experimental ART procedures and the number of eggs obtained, embryos donated for research, and the studies for which they were used. 4-6. Certification of clinics could allow greater control over any new ART procedures. The United Kingdom might be a model for certifying ART clinics, although the terms of the legislation would have to be adapted to the US federal style of government. REFERENCES 1. CENTERS FOR DISEASE CONTROL. 1998 Assisted Reproductive Technology Success Rates. 1998. National Summary and Fertility Clinic Reports. Online at: http://www.cdc.gov/nccdphp/drh/art.htm. 2. VANDERVORS M, STAESSEN C, SERMON K, DE VOS A, VAN DE VELDE H, VAN ASSCHE E, BONDUELLE M, VANDERFAELLIE A, LISSENS W, TOURNAYE H, DEVROEY P, VAN STEIRTEGHEM A, LIEBAERS I. The Brussels’ experience of more than 5 years of clinical preimplantation genetic diagnosis. Hum Reprod Update 2000 Jul-2000 Aug 31, 6(4):364-73. 3. Assisted reproductive technology in the United States and Canada: 1994 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 1996 Nov, 66(5):697-705. 4. CUNNINGHAM FG, MACDONALD P, GANT N, LEVENO KJ, GILSTRAP LC, HANKINS G, CLARK S: Williams Obstetrics. 20th Edition. McGraw-Hill; 1997:583. 5. LUNENFELD B, INSLER V. Classification of amenorrhoeic states and their treatment by ovulation induction. Clin Endocrinol (Oxf) 1974 Apr, 3(2):223-37. 6. NAVOT D, RELOU A, BIRKENFELD A, RABINOWITZ R, BRZEZINSKI A, MARGALIOTH EJ. Risk factors and prognostic variables in the ovarian hyperstimulation syndrome. Am J Obstet Gynecol 1988 Jul, 159(1):210-5. 7. TROUNSON AO, Monash University, Melbourne, Australia. Directed differentiation of embryonic stem cells and somatic cell nuclear transfer. Scientific and medical aspects of human cloning. National Academy of Sciences, Washington, D.C., 2001 Aug 7. Online at: www.nationalacademies.org/humancloning 8. GARDNER DK, LANE M, SCHOOLCRAFT WB. Culture and transfer of viable blastocysts: a feasible proposition for human IVF. Hum Reprod 2000 Dec, 15 Suppl 6:9-23. 9. TESARIK J, SOUSA M, GRECO E, MENDOZA C. Spermatids as gametes: Indications and limitations. Hum Reprod 1998 Jun, 13 Suppl 3:89-107; discussion 108-11. 10. FEIL R. Early-embryonic culture and manipulation could affect genomic imprinting. Trends Mol Med 2001 Jun, 7(6):245-6. 11. ARIEL M, CEDAR H, MCCARREY J. Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis. Nat Genet 1994 May, 7(1):59-63.

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Scientific and Medical of Aspects: Human Reproductive Cloning 12. KURINCZUK JJ, BOWER C. Birth defects in infants conceived by intracytoplasmic sperm injection: an alternative interpretation. BMJ 1997 Nov 15, 315(7118):1260-5; discussion 1265-6. 13. BOWEN JR, GIBSON FL, LESLIE GI, SAUNDERS DM. Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet 1998 May 23, 351(9115):1529-34. 14. BONDUELLE M, DEVROEY P, LIEBAERS I, VAN STEIRTEGHEM A. Commentary: Major defects are overestimated. BMJ 1997 Nov 15, 315:1265-66. 15. SUTCLIFFE AG, TAYLOR B, GRUDZINSKAS G, THORNTON S, LIEBERMAN B. Children conceived by intracytoplasmic sperm injection. Lancet 1998 Aug 15, 352(9127):578-9. 16. WENNERHOLM UB, BERGH C, HAMBERGER L, LUNDIN K, NILSSON L, WIKLAND M, KALLEN B. Incidence of congenital malformations in children born after ICSI. Hum Reprod 2000 Apr, 15(4):944-8. 17. BONDUELLE M, WILIKENS A, BUYSSE A, VAN ASSCHE E, WISANTO A, DEVROEY P, VAN STEIRTEGHEM AC, LIEBAERS I. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1996 Dec, 11 Suppl 4:131-55; discussion 156-9. 18. BONDUELLE M, CAMUS M, DE VOS A, STAESSEN C, TOURNAYE H, VAN ASSCHE E, VERHEYEN G, DEVROEY P, LIEBAERS I, VAN STEIRTEGHEM A. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod 1999 Sep, 14 Suppl 1:243-64. 19. WISANTO A, BONDUELLE M, CAMUS M, TOURNAYE H, MAGNUS M, LIEBAERS I, VAN STEIRTEGHEM A, DEVROEY P. Obstetric outcome of 904 pregnancies after intracytoplasmic sperm injection. Hum Reprod 1996 Dec, 11 Suppl 4:121-9; discussion 130. 20. WENNERHOLM UB, BERGH C, HAMBERGER L, WESTLANDER G, WIKLAND M, WOOD M. Obstetric outcome of pregnancies following ICSI, classified according to sperm origin and quality. Hum Reprod 2000 May, 15(5):1189-94. 21. SUTCLIFFE AG, TAYLOR B, SAUNDERS K, THORNTON S, LIEBERMAN BA, GRUDZINSKAS JG. Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case-control study. Lancet 2001 Jun 30, 357(9274):2080-4. 22. MANNING M, LISSENS W, BONDUELLE M, CAMUS M, DE RIJCKE M, LIEBAERS I, VAN STEIRTEGHEM A. Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod 2000 Nov, 6(11):1049-53. 23. IN’T VELD P, BRANDENBURG H, VERHOEFF A, DHONT M, LOS F. Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet 1995 Sep 16, 346(8977):773. 24. KENT-FIRST MG, KOL S, MUALLEM A, OFIR R, MANOR D, BLAZER S, FIRST N, ITSKOVITZ-ELDOR J. The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol Hum Reprod 1996 Dec, 2(12):943-50. 25. MESCHEDE D, LEMCKE B, EXELER JR, DE GEYTER C, BEHRE HM, NIESCHLAG E, HORST J. Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection—prevalence, types, sex distribution and reproductive relevance. Hum Reprod 1998 Mar, 13(3):576-82.

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