MODERN TRENDS. Edward E. Wallach, M.D. Associate Editor. Mark D. Johnson, M.D.

Transcription

1 FERTILITY AND STERILITY VOL. 70, NO. 3, SEPTEMBER 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. MODERN TRENDS Edward E. Wallach, M.D. Associate Editor Genetic risks of intracytoplasmic sperm injection in the treatment of male infertility: recommendations for genetic counseling and screening Mark D. Johnson, M.D. Division of Reproductive Endocrinology and Genetics, Department of Obstetrics and Gynecology, Harbor-University of California Los Angeles (UCLA) Medical Center, UCLA School of Medicine, Torrance, California Objective: To review the most clinically significant genetic disorders associated with severe oligospermia and azoospermia in males, and to present recommendations for the genetic counseling and screening of infertile males and their partners before undertaking intracytoplasmic sperm injection (ICSI)-assisted reproduction. Design: The literature on genetic disorders associated with severe oligospermia and azoospermia was reviewed, and the most recent outcome data from surveys of ICSI-derived offspring are presented. Studies related to this topic were identified through MEDLINE. Result(s): Genetic disorders are not infrequent causes of severe oligospermia and azoospermia in males undergoing ICSI-assisted reproduction. The application of ICSI in the treatment of oligospermic or azoospermic males may result in the transmission or de novo introduction of genetic mutations or chromosomal abnormalities in their offspring. Genetic counseling and appropriate screening of couples with male infertility should be performed before their undertaking ICSI-assisted reproduction. Conclusions: An understanding of the genetic risks and possible consequences that are inherent when ICSI is used to assist fertilization in couples with male infertility is necessary for clinicians and their patients. (Fertil Steril 1998;70: by American Society for Reproductive Medicine.) Key Words: Azoospermia, oligospermia, male infertility, intracytoplasmic sperm injection, chromosomal abnormality, cystic fibrosis, AZF, Kennedy s disease, genetic counseling Received November 10, 1997; revised and accepted April 13, Reprint Requests: Mark D. Johnson, M.D., Center for Reproductive Medicine and Genetics, 5500 Village Blvd., Suite 103, West Palm Beach, Florida (FAX: ) /98/$19.00 PII S (98)00209X Intracytoplasmic sperm injection (ICSI) is a highly effective therapeutic intervention used to assist fertilization with sperm from an infertile male, and its innovation offers a previously unavailable opportunity for couples with male factor infertility to now produce children. Its inception has revolutionized the management of male infertility. However, critics suggested that the use of ICSI for the treatment of male infertility has the potential to negatively impact the genetic composition of the human population for future generations (1, 2). These concerns have considerable merit when they are examined from the viewpoint that ICSI is a technique that bypasses the effective biologic mechanisms of sperm selection that were set in place during the evolution of the human reproductive process. The application of ICSI to human reproduction has not been preceded by an extensive research trial in other mammals. Consequently, the human experience with ICSI is the experimental record. The application of ICSI foregoes an understanding of the underlying etiology of the individual male s infertility, which may be of a transmissible genetic basis. Darwinian fitness is defined as the efficiency of producing offspring in comparison with the fertile norm (3). The use of ICSI to alter the reproductive performance of a male with infertility resulting from a specific genetic constitution or genotype changes the fitness of his genotype. Taken in this context, the application of ICSI in assisted reproductive technology provides the ability to dramatically improve the fitness of many mutant male genotypes and potentially alter the genetic composition of a population. It is therefore essential to have an understanding of the 397

2 genetic risks and possible consequences that are inherent when ICSI is used to assist fertilization in the population of infertile males. This overview presents the most clinically significant genetic disorders associated with male infertility and provides a genetic differential diagnosis for the infertile male that should be considered before initiating ICSI-assisted reproduction. To assist in the counseling and screening of couples with male factor infertility who are considering ICSI, recommendations are provided for the genetic counseling and screening of infertile males and their partners before undertaking ICSI-assisted reproduction. CHROMOSOMAL ABNORMALITIES AND MALE INFERTILITY A range of cytogenetic abnormalities is associated with disturbances of spermatogenesis resulting in impaired male fertility. A review of pooled data from 11 surveys of 9,766 infertile men with azoospermia and oligozoospermia (Table 1) revealed a 5.8% incidence of chromosomal abnormalities (4 13). Sex chromosome anomalies were predominant in azoospermic and oligospermic infertile men with an incidence of 4.2%, whereas autosome anomalies were observed in only 1.5% of the combined populations. As shown in Table 1, these frequencies were significantly higher than the 0.38% incidence of chromosome anomalies reported in a series of phenotypically normal newborn cytogenetic screens. The incidence of sex chromosome and autosome anomalies in newborn screens was 0.14% and 0.25%, respectively (13). In a recently published review of the literature (13), data were pooled from six cytogenetic surveys of azoospermic men and five cytogenetic surveys of oligozoospermic men (with sperm per ml) men. The cumulative data from the cytogenetic surveys of the azoospermic and oligozoospermic men revealed an incidence of cytogenetic abnormalities in 13.7% and 4.6% of the populations, respectively. Klinefelter s syndrome (47,XXY) and mosaic Klinefelter s (46,XY;47,XXY) were the predominant abnormalities observed in 10.8% of the azoospermic males. Non-Klinefelter sex chromosome anomalies and autosome anomalies were observed in only 1.8% and 1.1% of azoospermic males, respectively. In the oligozoospermic population, autosome anomalies were reported in 3% of the males, and sex chromosome anomalies were observed in only 1.6% of the population (13). Aneuploid Conditions: 47,XXY (Klinefelter s Syndrome) and 47,XYY Adult males with Klinefelter s syndrome have atrophic, hyalinized testes that are severely depleted or devoid of germ cells. The germ cell depletion appears to be secondary to a process of advanced germ cell atresia that is theorized to result from a lethal gene dosage from the increased number of X chromosomes in the testis environment( (14, 15). Males with 46,XY;47,XXY gonadal mosaicism have variable numbers of viable XY germ cells in their testes that may contribute to sperm production (16). Spermatozoa from azoospermic 47,XXY males have been used to fertilize oocytes by ICSI (17). Tournaye et al. (17) successfully retrieved a few rare testicular spermatozoa from testicular biopsies from three 47,XXY Klinefelter s men for ICSI fertilization. The sperm were injected into oocytes to produce five 6-cell stage embryos. All of the 6-cell embryos underwent blastomere biopsy and fluorescence in situ hubridization (FISH) analysis with X and Y probes, confirming a normal sex chromosome pattern in all five embryos (18). Unfortunately, only one biochemical pregnancy resulted from the transfer of four embryos into three recipients. Recently, Palermo et al. (19) have reported the successful births of three normal offspring from two pregnancies that were conceived by ICSI after testicular retrieval of sperm from 47,XXY men. The normal sex chromosome complement of the offspring indicates that sperm with a normal haploid complement of X or Y were retrieved from the testes of these Klinefelter s men. Haploid sperm could be produced from these men under two mechanisms: [1] if they have confined gonadal 46,XY/47,XXY mosaicism with XY germ cells residing in a small percentage of the tubules of their testes and not in the cells that comprise their bone marrow, or [2] that 47,XXY male germ cells are viable and capable of progressing through meiosis to produce normal haploid spermatozoa. The second mechanism is in direct contradiction to the established viewpoint that 47,XXY germ cells are lethal and undergo premature atresia and is the less likely explanation (16). Preimplantation FISH analysis of embryos for sex chromosome ploidy assessment is recommended following ICSIassisted fertilization with sperm recovered from Klinefelter s males (18). Future studies to determine sex chromosome ploidy of sperm from affected males will be helpful in determining the frequencies of normal haploid and hyperploid 24,XY-bearing sperm in the population of Klinefelter s males. 47,XYY Most of 47,XYY males are fertile and produce chromosomally normal 46,XY sons and 46,XX daughters (20). However, this population is variable in its phenotype and 47,XYY males may present with severely impaired sperm production (15, 21). The mechanism of reduced sperm production in affected individuals appears to be secondary to spermatogenic arrest of most of YY germ cells due to abnormal meiotic pairing during meiosis (22). It has been reported that the risk that a 47,XYY male will conceive aneuploid offspring is not increased above the background risk for the normal 46,XY male (20). The reproductive outcome from the use of ejaculated spermatozoa for ICSI from a 47,XYY male is anticipated to be similar to that of a 398 Johnson Genetic risks of ICSI in male fertility Vol. 70, No. 3, September 1998

3 TABLE 1 Number of chromosomal abnormalities in azoospermic and oligozoospermic infertile men compared with phenotypically normal newborns. Reference No. of males No. of sex chromosome abnormalities (%) No. of autosome abnormalities (%) Total no. of chromosome abnormalities (%) (4) 1, (2.7) 6 (0.06) 33 (3.3) (5) 2, (1.4) 18 (0.7) 51 (2.1) (6) (6.7) 10 (2.8) 34 (9.5) (7) 2, (6.9) 40 (1.6) 215 (8.6) (8) (1.8) 4 (1.2) 10 (2.9) (9) (4.1) 7 (2.2) 20 (6.3) (10) (5.5) 9 (1.1) 54 (6.6) (11) (5.0) 10 (2.8) 35 (7.0) (12) (6.8) 33 (3.4) 98 (10.3) (13) (0.35) 12 (2.1) 14 (2.4) Total 9, (4.2) 149 (1.5) 564 (5.8) Newborn infants (13) 94, (0.14) 232 (0.25) 366 (0.38) normal 46,XY male, but future clinical experience using ICSI with sperm from XYY men will be required to support this theoretical projection. X-Autosome and Y-Autosome Translocations Reciprocal X-autosome translocations uniformly cause male infertility, regardless of the position of the breakpoint in the X. Male heterozygotes present with severe spermatogenic arrest that frequently results in azoospermia (23). In reciprocal X-autosome translocations, a segment of the X chromosome containing X-genetic loci is translocated to an autosome, and the X-loci become linked to elements controlling autosomal gene expression. The translocated X- segment containing X-genetic loci is presumed to remain transcriptionally active because the X-inactivation elements have been removed from control of the translocated segment. The abnormal X-inactivation of the translocated segment interferes with the genetic control of germ cell progression, resulting in meiotic arrest at the primary spermatocyte stage (24). Reciprocal translocations involving Y chromosome segments may result in aberrant spermatogenesis due to abnormal sex chromosome pairing during meiosis, abnormal testis determination in the indifferent gonad (impaired SRY), or defective transcription of the Y-chromosomal azoospermia factor(s) (AZF), which is discussed in Yq11 Deletions and the Azoospermia Factors (25, 26). The phenotype is variable and depends on the location of the breakpoints of the translocated segments and the degree of disrupted sex chromosome pairing during meiosis that results in sperm maturation arrest. Reciprocal Autosomal Translocations The association of reciprocal autosomal translocations with male sterility was first reported by Lyon and Meredith (27) in their observation of abnormal behavior of the rearranged autosomes in meiosis during spermatogenesis in mice. The translocated chromosome segments were observed to attempt homologous pairing unsuccessfully, leaving large unpaired segments free to interfere with the X and Y chromosomes during meiosis I. Abnormal pairing between the unpaired autosome segments and the X interferes with the normal X inactivation during spermatogenesis, resulting in a a lethal gene dosage effect on the male germ cells (28). Similar abnormal meiotic mechanisms occur during spermatogenesis in males who are heterozygote carriers of reciprocal autosomal translocations. Increased frequencies of reciprocal autosomal translocations have been reported in azoospermic (0.9%) and severely oligozoospermic (0.6%) men compared to the incidence in phenotypically normal newborns (0.1%) (13). The D-group [13, 14, 15] and G- group [21, 22] acrocentric chromosomes are most frequently involved in the rearrangements that are associated with failed meiotic pairing resulting in male infertility (15, 29). Robertsonian Translocations An increased number of carriers of Robertsonian translocations have been reported among severely oligozoospermic (1.6%) and azoospermic (0.09%) men attending infertility clinics compared with the incidence in phenotypically normal newborns (0.08%) (13). The most common Robertsonian translocation observed in the male infertility population is the t(13q14q). Meiotic studies of infertile carriers of 13q14q and 14q21q Robertsonian translocations reveal abnormal behavior of the rearranged autosomes in meiosis during spermatogenesis (30, 31). Most t(13q14q) cases are familial, and many of the infertile carriers have fertile relatives carrying the same rearrangement. The reason for the occasional infertility in generations is not known; however, selected transmission of molecular abnormalities associated with the translocation may occur, or genetic background may have an influence (15). FERTILITY & STERILITY 399

4 Risks of ICSI for Male Carriers of Chromosomal Translocations Balanced translocation carriers, either reciprocal or Robertsonian, do not necessarily have infertility or abnormal phenotypes. However, because translocations interfere with normal chromosome pairing and segregation at meiosis I, there is potential for formation of unbalanced gametes and subsequent unbalanced abnormal offspring. In carriers of balanced translocations, spermatogenesis results in the production of three combinations of normal, abnormal balanced, and abnormal unbalanced gametes. The first two combinations can lead to phenotypically normal offspring; however, the latter combination may produce unbalanced conceptions containing a duplication and a deletion of segments of chromosomes (32). The actual risk of abnormal gamete production faced by azoospermic or severely oligozoospermic carriers of balanced translocations is difficult to quantify before fertilization. The sizes of the chromosome segments, the chromosome breakpoint locations, and the type of rearrangement are critical factors in the meiotic pairing and chromosome segregation process. Although a considerable number of aneuploid conceptions derived from abnormal sperm will be spontaneously aborted or undergo fetal demise, infertile carriers of balanced translocations who undergo ICSI have a significant risk of producing a liveborn infant with chromosome imbalance. Baschat et al. (33) reported a pregnancy of a male fetus with an unbalanced 22;Y translocation derived from ICSI with sperm from an oligoasthenoteratozoospermic male with a 46,X,t(22;Y)(p11;q12) karyotype. Production of normal, balanced, and unbalanced gametes were all possible from this male, and in this circumstance, the fetus received an unbalanced translocation, which is a much more serious condition than his father s genotype. If the fetus had extraneous or deleted material from chromosome 22, it would be at risk to be affected with one of the serious syndromes associated with chromosome 22 abnormalities. This case illustrates the absolute necessity for cytogenetic screening of all infertile males considering ICSI treatment. Testart et al. (34) reported the results of their program of karyotype screening all couples with severe male infertility and their ICSI-derived offspring. In the 261 couples treated, 11 males (4.2%) and 3 females (1.2%) had abnormal karyotypes, all consisting of structural chromosome anomalies. The 11 chromosomally abnormal males had six Robertsonian 13q14q translocations, two autosomal translocations, two autosomal inversions, and one marker chromosome. In the 83 ICSI-conceived pregnancies, eight fetuses were produced from the 14 couples with carriers of chromosome structural anomalies. Three of the eight offspring were normal, and five fetuses inherited the identical chromosomal abnormalities found in their parents. Testart et al. (34) concluded that chromosomally normal offspring can be conceived through ICSI from infertile carriers of balanced chromosome translocations. In addition, the investigators strongly recommended that karyotypes be performed on all infertile men before initiating ICSI, and prenatal diagnosis should be offered for all ICSI-derived pregnancies (34). Inversions Autosome inversions are not generally associated with impairment of spermatogenesis, except for pericentric inversions of chromosome 1. Chandley et al. (35) reported the observation of meiotic pairing disturbances manifested by failed synapses formation during meiosis I in heterozygote carriers of In(1), resulting in male germ cell meiotic arrest. Supernumerary Marker Chromosomes and Rings Carriers of marker chromosomes and ring chromosomes are at risk for spermatogenic impairment due to meiotic arrest and instability that frequently results in maturation arrest at the spermatocyte stage (15). Yq11 Deletions and the AZFs Some infertile men with azoospermia or severe oligoasthenospermia have associated abnormalities of the Y chromosome. In 1976, Tiepolo and Zuffardi (36) described six azoospermic men with a distal deletion of Yq11. On the basis of these six azoospermic men, as well as two cases of ring Y chromosome with loss of Yq12 and part of Yq11, Tiepolo and Zuffardi proposed the presence of spermatogenesis-controlling factors at the distal portion of Yq11. Numerous reports of men with similar cytogenetic deletions of distal Yq11 and azoospermia or severe oligoasthenospermia have appeared in the literature since the initial report, supporting the hypothesis of the presence of spermatogenesis regulating genes within the distal Yq11 region (37, 38). Strong molecular evidence for the presence of spermatogenesis regulating genes on the human Y chromosome was provided by the detection of microdeletions within interval 6 in the distal Yq11 region in azoospermic men (26, 39 43). These preliminary studies have led to several gene searches to identify the specific AZF(s) that reside within distal Yq11. A comprehensive physical map of the Y chromosome was developed that incorporated more than 100 sequence tagged sites (STS) throughout the entire chromosome (44, 45). Since the development of this physical map, Y-linked sequence tagged sites have been used to screen DNAs derived from azoospermic or severely oligozoospermic males, in a further effort to identify genes and characterize deletions in the AZF region. The finding of overlapping interstitial Yq deletions in three azoospermic males led Ma et al. (42) to the identification of two closely related genes, YRRM1 and YRRM2, whose absence was proposed by them to cause azoospermia. Since the identification of YRRM1 and YRRM2 as possible candidates for the AZF(s), Reijo et al. (46) identified 400 Johnson Genetic risks of ICSI in male fertility Vol. 70, No. 3, September 1998

5 FIGURE 1 A diagramatic map of the human Y chromosome with the approximate sites of the repetitive YRRM1 and YRRM2 gene sequences and the DAZ gene within the AZF region. The SRY, RPS4Y, and ZFY loci are located away from the AZF region on Yp. another AZF candidate gene whose absence is associated with azoospermia, the deleted in azoospermia (DAZ) gene. Members from at least two gene families lie in the distal Yq11 region containing the AZF locus, namely YRRM (RBM) and DAZ (Figure 1). Linkage is not apparent between YRRM (RBM) and DAZ because deletions of one gene are not always associated with deletion of the other gene. In the process of their molecular scanning of the Y in 89 men with nonobstructive azoospermia, Reijo et al. (46) found 12 men with microdeletions for DAZ, but none were deleted for the YRRM genes. In contrast, Najmabadi et al. (47), Pryor et al. (48), and Kent-First et al. (49) reported in their screenings of infertile men that many of the deletions in AZF associated with azoospermia did not involve DAZ. Although DAZ is not a member of the YRRM (RBM) gene family, there appear to be significant similarities in molecular structure and function between the two gene families. Both DAZ and YRRM (RBM) encode RNA binding domains and are likely to function by binding RNA or single stranded DNA. Second, both the DAZ and YRRM (RBM) coding sequences contain a series of dissimilar tandem repeats. The DAZ and YRRM (RBM) genes both reside in several regions of the Y that contain an abundance of Y- specific repetitive sequences. DAZ and YRRM (RBM) are expressed specifically in the testis. It is highly probable that testis-specific RNA-binding proteins encoded by DAZ and YRRM (RBM) function during spermatogenesis (46). Azoospermic men with microdeletions of AZF exhibit a wide spectrum of spermatogenic defects, ranging from the complete absence of germ cells (Sertoli cell only syndrome) to meiotic maturation arrest with the occasional rare production of mature spermatids and morphologically abnormal sperm variants. Large deletions involving the AZF locus generally produce azoospermia; however, attempts to correlate the size of the microdeletions involving DAZ or the YRRM (RBM) genes with a specific phenotype or severity of spermatogenic impairment (Sertoli cell only syndrome or maturation arrest) have not always been accurate (46, 49). The frequency of microdeletions of the AZF locus in the azoospermic and severely oligozoospermic ICSI population ranges from less than 9% to 18% (46, 49). Deletions of AZF appear to arise de novo at a relatively high frequency in the general population. Reijo et al. (46) calculated that at least 1 in 10,000 male newborns carries a deletion in the AZF region that is not detectable in their father s intact Y. The mechanism(s) of how these microdeletions in the AZF region of the Y appear de novo have not been clearly resolved; however, recent studies by Kent-First et al. (49) have proposed some interesting hypotheses. Kent-First et al. (49) investigated the frequency of AZF microdeletions in 32 pairs of infertile fathers and their sons who were conceived with use of ICSI. They found microdeletions within the AZF region in three of the father/son pairs; one microdeletion was detected in both father and son, and two microdeletions were detected only in the sons and not in their respective fathers or the fertile controls. An AZF microdeletion of a 2.5-kb DNA segment between the YRRM (RBM) and DAZ loci was detected in the leukocyte DNA segments from one father and son pair. Although microdeletions in AZF were not detected in the leukocyte DNA segments from the other two fathers, their respective sons displayed distinct AZF microdeletions in their leukocyte DNA segments. One of the microdeletions that was detected only in the son and not in the father was relatively small in size, located between the YRRM (RBM) and DAZ loci. The other microdeletion detected only in the son s DNA was significantly larger than 2.5 kb and involved several consecutive STS and a deletion of both YRRM (RBM) and DAZ loci. Because two of the reported deletions do not include loss of the YRRM (RBM) or DAZ loci, other yet uncharacterized AZF spermatogenesis-related genes may reside within the DNA segment between the two loci (49). Microdeletions near or within the AZF region may occur as pre-meiotic or post-meiotic de novo germ line mutations in a fertile male (49). During spermatogenesis, aberrant pairing and recombination may occur between the X and Y chromosomes outside of the pseudoautosomal region in DNA segments of similar homology or similar repeat sequences, such as Yq (50). These mutation events that occur during spermatogenesis result in the production of mutant FERTILITY & STERILITY 401

6 sperm that function to produce a male with AZF microdeletions in his germ line and in all of his somatic cells, including his bone marrow. Microdeletions near or within the AZF region may also occur as post-zygotic de novo events resulting in an infertile individual who is mosaic for cell lines colonized with mutant Y chromosomes and intact Y chromosomes. Kent-First et al. (49) hypothesized that a significant number of azoospermic and severly oligozoospermic males may have gonadal mosaicism for AZF microdeletions. The investigators suggested that in some infertile men, AZF microdeletions are prevalent in a percentage of the cells that comprise their germ lines, but the mutations are not present in the cells that comprise their bone marrow (49). This hypothesis would explain the difference in AZF microdeletion detection in the two father/son pairs in which the sons were found to carry the microdeletions in their leukocyte DNA samples but the fathers did not. However, an infertile male who carries the AZF microdeletion in only a fraction of his spermatogonia may father normal sons who carry intact Y chromosomes. This hypothesis would also explain the variability of histologic spermatogenic impairment, including the tubule to tubule variability that is frequently observed in the testes from some of these infertile males. MEIOTIC ABNORMALITIES, SPERM ANEUPLOIDY, AND INFERTILITY Meiotic abnormalities that result in maturation arrest at different stages of meiotic division have been reported in cytogenetically normal infertile men (51 53). The reported meiotic abnormalities include asynapsis, desynapsis, low chiasma frequency, bivalent fragmentation, and asymmetric bivalents resulting in spermatogenic arrest and subsequent infertility (51, 52). Martin (53) has proposed that most 46,XY infertile males are affected by abnormal meiotic pairing in both sex chromosomes and autosomes, resulting in an interruption of normal spermatogenesis that ultimately results in decreased sperm production and an increased frequency of aneuploid spermatozoa. Cytogenetic Analyses of Spermatozoa from Infertile Males The frequency of chromosomal abnormalities in spermatozoa from the normal male population as determined by hamster ovum fertilization and cytogenetic analysis is approximately 10% (13, 54 56). Guttenbach et al. (57, 58) directly screened 110,000 sperm cells from 20 fertile men, aged years, using FISH with chromosome-specific DNA probes for chromosomes 3, 7, 10, 11, 17, 18, X and Y. The frequency of meiotic nondisjunction remained consistantly similar for each autosome and sex chromosome. The rate of disomy for any individual chromosome in sperm cells was estimated to be between 0.31% and 0.41%, and remained relatively unchanged for each chromosome in the sperm from fertile men within the age range (57, 58). Recent studies using FISH analyses of different chromosomes directly in spermatozoa nuclei from infertile males revealed an increased frequency of aneuploidy (53, 59, 60). Pang et al. (abstract) used FISH probes for 7, 11, 12, 18, X and Y to analyze chromosome number in the spermatozoa from nine oligoasthenoteratozoospermic (OAT) males. They observed a 19.6% frequency of autosome and sex chromosome diploidy and nullisomy in the infertile males with OAT compared to a 1.45% frequency in fertile controls. Since this ploidy analysis was limited only to a partial genome screening of the sex chromosomes with four autosomes and did not screen the sperm genome for all of the 22 autosomes, the true frequency of aneuploidy in the genomes of these sperm may be significantly higher than 19.6%. Bernardini et al. (59) performed an analysis of sex chromosome ploidy by in situ hybridization in sperm from nine OAT males and six couples with unexplained infertility and observed a significant increase in sex chromosome aneuploidy in the sperm from the males with OAT when compared to men with normal semen parameters. Moosani et al. (60) performed sperm karyotyping of 518 human sperm-hamster oocyte fusion products derived from five infertile men with abnormal sperm parameters, and reported a 3.1% frequency of aneuploidy in the sperm from infertile men compared with the 0.84% aneuploidy in the sperm from fertile males. The investigators expanded their analysis and used FISH probes for 1, 12, X, and Y combined with hamster ovum penetration cytogenetics to analyze sperm genotypes in ten oligospermic, asthenospermic, or teratozoospermic 46,XY infertile men. They observed an increased frequency of disomy for chromosome 1 and the sex chromosomes in the infertile men compared to normal fertile controls (60). These studies of infertile males suggest a process of abnormal pairing in both sex chromosomes and autosomes that interrupts normal spermatogenesis, resulting in severely decreased sperm production and an increased frequency of aneuploid spermatozoa (53). Recently, molecular cytogenetic analyses have begun to correlate some specific morphologic abnormalities of sperm with chromosomal abnormalities. A chromosome analysis of spermatozoa with enlarged head size and multiple tails produced from men with OAT correlated abnormal morphology with polyploidy. Molecular cytogenetic FISH analysis of sperm cells with probes specific for chromosomes X, Y, and 18 revealed virtually all sperm cells to be aneuploid with 40% diploidy and 24% triploidy frequencies (61). This correlation of sperm with enlarged heads and multiple tails with a polyploidic genotype is well established in the mouse (62). The condition appears to arise by omission of one meiotic division during spermatogenesis, resulting in polyploid spermatozoa. A novel approach for sperm genotyping has been described in which human sperm cells are microinjected into 402 Johnson Genetic risks of ICSI in male fertility Vol. 70, No. 3, September 1998

7 mouse oocytes followed by cytogenetic analysis of the hybrid zygote (63). Spermatozoa with normal head morphology had a 1.3% incidence of aneuploidy and a 6.9% incidence of structural chromosomal abnormalities. Preliminary results suggest that some morphologic abnormalities, specifically spermatozoa with amorphous, round and elongated heads, are associated with an increased frequency (26.1%) of structural chromosomal abnormalities when compared with the incidence in morphologically normal spermatozoa. The results of these studies caution against the injection of spermatozoa that appear abnormal into human oocytes during ICSI (63). Cytogenetic Results of ICSI-Derived Offspring from Infertile Males Because the proportion of spermatozoa with abnormal chromosome complement is increased in men with severely abnormal sperm parameters (OAT) or nonobstructive azoospermia, the possibility that infertile fathers who undergo ICSI will transmit chromosomal abnormalities to their offspring is a major concern. The initial published reports from prenatal diagnoses performed on pregnancies conceived from infertile men through ICSI suggested a predisposition to the production of offspring with sex chromosome aneuploidy (64 66). Based on the evidence accumulated from the most recent genetic surveys of obstetric outcomes (67) and data from children who are conceived through ICSI (68), it appears that offspring conceived through ICSI for the treatment of male factor infertility appear to have a slightly increased risk for sex chromosome aneuploidy than children who are not. Prenatal screening was performed in 585 of the pregnancies, the majority by second trimester amniocentesis (67, 68). Fetal screening revealed 12 (2%) abnormal karyotypes, 6 (1%) de novo abnormalities, and 6 (1%) structural abnormalities paternally transmitted directly from the father s karyotype. The six cases of de novo abnormalities were: 47,XYY (maternal age 25), 47XXX (maternal age 32), two fetuses with 47,XXY (maternal ages 28), a 46XX/47XXX mosaic (maternal age 44), and a fetus with trisomy 21 (maternal age 41). Although chromosomal analyses to determine the parent of origin were not performed to ascertain the etiology of the extra chromosomes, the trisomy 21 error is presumably secondary to maternal nondisjunction, whereas the others have a strong likelihood of paternal chromosome etiology. The 46XX/47XXX mosaic resulted from a postmeiotic error during early cleavage in a zygote that has a high probability of being originally trisomic (47,XXX) rather than 46,XX (69). The 1% incidence of de novo sex chromosome aneuploidy observed in this survey is significantly higher than the 0.14% to 0.19% incidence observed in non-icsi conceived newborns (13, 70). The slight increase in de novo sex chromosome aneuploidy is suggested by the investigators to be secondary to the increased frequency of sperm aneuploidy observed in infertile men, rather than a result of the ICSI technique (68). Perrson et al. (71) suggested that the increased frequency of sex chromosome anomalies in ICSI-assisted offspring is the result of using the diploid sperm from previously unidentified Klinefelter mosaic males. Perrson et al. (71) strongly suggested karyotyping all males before they undergoing ICSI to categorize individuals into low risk aneuploid or high risk aneuploid groups. Martin (53) proposed that most 46,XY infertile males are predisposed to abnormal pairing in both the sex chromosomes and autosomes during meiosis, resulting in decreased sperm production and an increased frequency of aneuploid spermatozoa. Martin (53) suggested that karyotyping males to identify those at risk for producing aneuploid offspring is not helpful. However, she recommended that all couples who achieve a pregnancy after undergoing ICSI be offered prenatal genetic diagnosis (53). First Trimester Losses/Major Congenital Malformation Rates in ICSI-Derived Pregnancies The obstetric outcomes of 1,447 pregnancies resulting in 1,455 children conceived after ICSI have been compiled from the largest American and European published surveys to date (67, 68, 72). The incidences of first trimester losses in pregnancies conceived through ejaculated spermatozoa, epididymal spermatozoa, and testicular spermatozoa were 24.6%, 31.2%, and 33.3%, respectively. No differences were observed in the obstetric outcomes of the children conceived through ICSI with sperm from the different sources (67). In the 1,455 children born following ICSI, 32 (2.2%) had a variety of major congenital malformations, none of which were disproportionately frequent. The 2.2% observed incidence is within the normally expected range of congenital malformations. Genetic Counseling, Karyotype Screening, and AZF Microdeletion Testing Because infertile males have a higher risk than the normal male population of carrying and thus transmitting to their offspring significant chromosomal abnormalities, karotyping of peripheral blood should be routinely performed for these individuals before proceeding with ICSI; the results of these blood tests can then be used by genetic counselors. Before initiating ICSI, azoospermic and oligospermic males with chromosomal abnormalities should be identified, and the couple should receive nondirective, objective genetic counseling regarding the nature of the chromosomal abnormality and their potential risks for abnormal offspring. If the infertile male is determined to be a carrier of a chromosomal abnormality by peripheral blood karyotype, then the couple should be counseled by a certified genetics counselor and/or a clinical/prenatal geneticist on the reproductive risks associated with the affected male s specific abnormality. Counseling should be nondirective and should FERTILITY & STERILITY 403

8 provide the couple with the necessary information to decide if they wish to proceed with ICSI-IVF and possible future prenatal genetic testing, or if they wish to consider other reproductive options. Counseling should be based on the following guidelines: 1. If the male is a carrier of a balanced autosomal abnormality, the couple should be informed that the pregnancy success rate may be decreased, and that an increased risk for miscarriage exists. The couple should be counseled that their fetus may be: [a] affected with a chromosomal abnormality that may cause multiple congenital anomalies, mental retardation, and a reduced life expectancy, or [b] their fetus may be normal, or [c] their fetus may carry the same chromosomal abnormality as the father, resulting in reduced fertility or infertility. 2. If the male is a carrier of a structural abnormality involving only the sex chromosomes, there is an increased risk of transmitting infertility and possibly other disorders or malformations, depending on the location of the breakpoint. 3. If the male has sex chromosome aneuploidy (47,XXY, etc.), the couple should be counseled that the success rate of ICSI is variable, there may be an increased risk for miscarriage, and there is a risk of transmitting sex chromosome aneuploidy to the offspring. 4. Prenatal diagnosis of the ICSI-conceived pregnancy should be offered to the couple based on the chromosomal abnormality in the male. 5. The couple should be informed that preimplantation diagnostic screening of their preembryos by FISH may be a consideration for them, even if this highly specialized diagnostic method is not available as an option for them locally. 6. Finally, the couple should be offered the possible alternative of donor insemination in lieu of ICSI-assisted fertilization. Infertile 46,XY men considering ICSI should receive genetic counseling regarding their risks of having a microdeletion in AZF that could be the cause of their infertility and that the microdeletion could be transmitted to their sons, probably resulting in their future infertility. Couples should be offered the opportunity to be screened for microdeletions in the AZF region. Infertile nonmosaic men who carry AZF microdeletions in their leukocyte DNA will transmit the mutation to all of their sons. If gonadal mosaicism is prevalent in the selected infertile male population, then each individual s risk of transmitting a microdeletion is greater than the generally quoted incidence of 18%. The transmission of mutations in men with gonadal mosaicism depends on the ratio of intact Y to mutant Y chromosomes in his germ line. Kent-First et al. (49) proposed that the management of infertile males with possible AZF microdeletion gonadal mosaicism would be improved through preimplantation genetic diagnostics. Preimplantation diagnostics would allow for separation of ICSI generated embryos carrying AZF microdeletions from embryos with intact Y chromosomes before transfer of the unaffected embryo(s). Couples in which 46,XY infertile men are candidates for ICSI should also receive genetic counseling that is based on the evidence accumulated to date, ICSI-derived offspring from couples with male factor infertility have a slightly increased incidence of sex chromosome aneuploidy (1%) in comparison to the incidence (0.14% to 0.19%) in the non- ICSI-derived newborn population (13, 53, 59, 60, 64 68, 70). The frequency of sex chromosome aneuploidy (1%) in a large prenatal cytogenetic survey of pregnancies conceived from ICSI is higher than the generally accepted loss rate for second trimester amniocentesis (0.5%). It is reasonable to offer prenatal diagnosis as an option to couples with male factor infertility during their ICSI-conceived pregnancies. Couples should be reassured that there is no evidence to suggest that ICSI predisposes to an increased risk of conceiving offspring affected with: [1] other chromosome anomalies or [2] congenital malformations, unless other parental factors (i.e., abnormal karyotype or family history) predispose to such abnormalities (67, 68, 72). CYSTIC FIBROSIS AND MALE INFERTILITY Cystic fibrosis (CF) is one of the most common genetic disorders affecting Caucasians of European descent (73). It is an autosomal recessive disease affecting approximately 1 in 2,500 children with a carrier frequency in the Caucasian population of 1 in 25. CF is caused by mutations in the gene of the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that regulates transport of electrolytes across epithelial-cell membranes (73). To date, more than 600 mutations and DNA sequence variations have been identified in the CFTR gene (74). The classic clinical manifestations of the disease are progressive obstruction and infection of the respiratory tract, pancreatic exocrine deficiency, clinically demonstrable sweat abnormality, and in affected males, obstructive azoospermia (75). More than 95% of males with CF are infertile secondary to obstructive azoospermia (73). The obstructive azoospermia is caused by maldevelopment of the mesonephric ducts, resulting in either agenesis or atresia of the epididymis, vas deferens and/or seminal vesicles. It is not known whether the mesonephric maldevelopment resulting in the congenital bilateral absence of the vas deferens (CBAVD) is a primary developmental anomaly or if it is a secondary degenerative change resulting from abnormal mucus secretion with chronic ductal obstruction (73, 76). CBAVD presents in nearly all CF males irrespective of the severity of their pancreatic or pulmonary disease and may present as an isolated clinical manifestation or a specific genital phenotype. Affected individuals with CBAVD may have relatively normal pulmonary and pancreatic functions with normal or mildly elevated sweat electrolyte concentrations (75). Males presenting with isolated CBAVD carry increased frequencies of CFTR mutations when compared to fertile 404 Johnson Genetic risks of ICSI in male fertility Vol. 70, No. 3, September 1998

9 males and are either compound heterozygotes or carriers of a CFTR mutation and a unique intron 8 5T splice variant (76, 77). An extensive analysis of the entire coding region of the CFTR gene in 67 men with CBAVD revealed that 24% of patients were compound heterozygotes, and another 42% were carriers of one CFTR mutation, resulting in a 66% CF mutation carrier frequency (78). In a summary of data from 12 studies, Lissens et al. (76) reported that from a total of 420 men with CBAVD, 19% were found to be compound heterozygotes for CFTR mutations and 47% were carriers of a single CFTR mutation. These cumulative data estimate that 66% of men presenting with CBAVD will carry one or more CFTR mutations and that 34% of men with CBAVD have acquired this anomaly secondary to other developmental etiologies that are not related to CF (76). Most of the CFTR mutations associated with CBAVD have been previously described in patients with pulmonary and/or pancreatic CF, but some of the mutations or DNA sequence variations (intron 8 5T) have been exclusively identified in men with CBAVD. The intron 8 5T splice variant is a DNA sequence variation characterized by the number of thymidine (T) nucleotides in the splice acceptor site of the intron 8-exon 9 border. In the population, five, seven, or nine T nucleotides are found at this location. The presence of only 5 T nucleotides allows for a splicing abnormality with a loss of exon 9 from CFTR mrna, resulting in a nonfunctional CFTR protein (79). The intron 8 5T splice variant has been found in 46% of men with CBAVD but is not associated with the classic pulmonary or pancreatic manifestations of CF (77). In the compound heterozygotes, none of the patients had two severe phenotype mutations. The two alleles were frequently observed to be a compound severe phenotype mutation (deltaf508, etc.) and a mild phenotype mutation (R117H, etc.) or two mild phenotype mutations suggesting that CBAVD is a mild form of CF (76). Males presenting with CBAVD and unilateral renal malformations have a distinct clinical entity that does not represent a phenotype of CF (80). CF patients have normal ureters and kidneys. In contrast, these CBAVD patients frequently suffer renal agenesis and have normal CF sweat tests without CFTR gene mutations. They appear to have a non-cf related developmental anomaly of the Wolffian and mesonephric ducts (76, 80). Males with congenital unilateral absence of the vas deferens (CUAVD) may be normally fertile and therefore remain undiagnosed in the general population. The etiology of CUAVD is heterogeneous, and the affected population is comprised of two distinct subgroups based on vasal patency as well as CF mutation status (81). The subgroup of CUAVD males with anatomically complete and patent vas deferens on the contralateral side opposite to the vasal aplasia has no association with CFTR gene mutations, but has an incidence of ipsilateral renal malformations in 38%. Another subgroup, defined as having bilateral vasal abnormalities with single vas occlusion at the inguinal or pelvic level, is associated with CF mutations (81). In addition to its role in Wolffian and mesonephric duct development during early fetal life, the CFTR protein may be critically involved in the process of spermatogenesis. Increased frequencies of CF mutations have been observed in populations of healthy infertile men with abnormal sperm parameters and/or nonobstructive azoospermia. Van der Ven et al. (82) screened 101 healthy infertile men with abnormal sperm parameters for 13 CFTR gene mutations. They found that 14 of 80 (17.5%) of men with abnormal sperm parameters and 3 of 21 nonobstructive azoospermic males (14.3%) had at least one CFTR mutation (one azoospermic male was a compound heterozygote). This increased frequency of CFTR mutations in the selected populations of healthy infertile men (carrier rate of 16.8%) shows that these men are at significantly greater genetic risk than the normal U.S. population (carrier rate of 1.4% to 4%) for transmission of the CFTR mutation (73, 74, 82). Presently, greater than 600 CFTR mutations and DNA sequence variations have been identified, and commercially available tests generally screen for the mutations that are responsible for greater than 90% of the disease (75). Van der ven et al. (82) screened with a panel limited to only 13 mutations, and therefore their estimates of CF mutation carrier frequency may be conservative. CFTR gene mutations may be more prevalent in the healthy oligospermic and nonobstructive azoospermic male population than the 16.8% carrier frequency detected in the 13-mutation screen. Genetic Counseling and Screening of Infertile Males for CF Mutations The high incidence (66%) of CF mutations in individuals with CBAVD has led to the proposal for the routine CF screening of all males with obstructive azoospermia or CBAVD before ICSI (71, 75, 76). In view of the possibly conservative 16.8% CF mutation carrier frequency in the healthy infertile male population, screening for CF mutations should be strongly recommended for all infertile males before undergoing ICSI. If the male is positive for a CF mutation in his screening, then the couple should receive genetic counseling by a certified genetic counselor and/or a clinical or prenatal geneticist. The female partner should be offered CF mutation testing before ICSI, and if she is positive for a CF mutation, the couple should be counseled regarding the findings and the potential risks of producing a CF-affected offspring before ICSI. Preimplantation diagnostics and/or prenatal diagnosis should be offered for CF screening of offspring. In addition to this expanded recommendation for CF screening of all infertile males before initiating ICSI, the FERTILITY & STERILITY 405

10 National Institutes of Health (NIH) has issued a Consensus Statement on Genetic Testing for Cystic Fibrosis from a meeting of a panel of experts that met in April 1997 (74). The recommendations from the NIH Consensus Statement clearly outline the importance of providing accurate genetic counseling to all couples considering pregnancy and the necessity of offering CF testing to them: Counseling services must be accurate and provide balanced information to afford individuals the opportunity to make autonomous decisions....thepanel recommends offering CF genetic testing to adults with a positive family history of CF, to partners of people with CF, to couples currently planning a pregnancy, and to couples seeking prenatal testing (74). The importance of screening for CF mutations by commercial genotyping with a test panel that is adequate for detection of most mutations in a selected population cannot be overstressed. Discussions with the commercial laboratory may be necessary to add the intron 8 5T test to the panel because most screening panels for CF mutation testing do not presently include the 5T test. Single CFTR gene mutations and CF compound heterozygosity can be missed if the test panel is not adequate for screening the most prevalent mutations in the infertile male population (75). ANDROGEN RECEPTOR MUTATIONS AND MALE INFERTILITY: THE INFERTILE MALE SYNDROME AND KENNEDY S DISEASE Incomplete Androgen Insensitivity Variants and the Infertile Male Syndrome The range of phenotypes in individuals with defects in androgen receptor function varies from the 46,XY sex-reversed female with the complete androgen insensitivity syndrome, to the phenotypically normal, infertile male with severe oligospermia or azoospermia. Infertility is often seen in males with incomplete or partial androgen insensitivity manifested by micropenis, hypospadias, and/or cryptorchidism, but the prevalence of androgen receptor defects in phenotypically normal infertile men is not yet known (83). Aiman et al. (84) initially proposed that some phenotypically normal, infertile males may present as a phenotypic variant within the broad spectrum of androgen insensitivity. Their proposal was based on their studies of three unrelated, phenotypically normal, infertile men (two were azoospermic and one was severely oligospermic) with deficiencies of the androgen receptor and elevated serum gonadotropins and plasma testosterone production. In their follow-up evaluation of 28 unrelated phenotypically normal men with azoospermia or oligospermia, Aiman and Griffin (85) concluded that androgen resistance may be the cause of a significant fraction (40% or more) of idiopathic male infertility due to azoospermia or severe oligospermia, and that the defect may be manifested predominantly in the testes. This high prevalence of androgen resistance in phenotypically normal, infertile males reported by Aiman and Griffin (84, 85) was disputed by Bouchard et al. (86) in their reported studies on 24 men with idiopathic severe oligospermia and normal levels of androgen receptor. Morrow et al. (87) studied a similar population of 21 phenotypically normal, severe oligospermic males, and reported that 4 (19%) of their subjects had low levels of androgen receptor. These clinical reports suggested that defects in androgen receptor function, manifesting as androgen resistance in otherwise normal males, may result in impaired spermatogenesis and infertility. Recent investigations have provided new insights into the prevalence of androgen receptor mutations as an etiology of infertility in otherwise normal males (88 92). Van Roijen et al. (88) studied androgen receptor immunostaining in testicular tissue samples from 37 oligozoospermic men with idiopathic infertility. They concluded that immunoexpression of the androgen receptor was unrelated to the condition of the spermatogenic epithelium in their oligozoospermic men, and that inappropriate expression of the androgen receptor was not a cause or consequence of the idiopathic infertility in their oligozoospermic patients (88). Since publication of the Lubahn et al. (89) study, which described the isolation and cloning of the gene for the androgen receptor (AR) gene, a few studies have screened infertile males for AR gene rearrangements or mutations. Akin et al. (90) provided the first molecular evidence of an abnormality in the AR gene as a possible cause of impaired spermatogenesis. A deletion of the entire exon 4 of the AR gene was identified in one of seven azoospermic, otherwise normal, males. Exon 4 encodes for the hinge between the DNA and steroid binding domains that contains the hormone-independent nuclear translocation signal. The deletion of exon 4 from the androgen receptor gene in this azoospermic subject presumably results in decreased levels of nuclear translocation of the androgen receptor that are adequate for virilization, but inadequate for spermatogenesis. In a follow-up study, molecular scanning of the AR gene for deletions in 16 more oligospermic or azoospermic men with idiopathic infertility did not reveal any affected individuals (91). Yong et al. (92) described an infertile OAT male with a point mutation in the hormone binding domain of the AR gene who successfully responded to androgen therapy with increased sperm production and subsequent fertility. Ng et al. (abstract), from this same investigative group, have recently reported more infertile males with point mutations in the hormone binding domain of the AR gene. To date, the prevalence of the AR gene mutations responsible for impaired spermatogenesis in phenotypically normal infertile men has not been definitively established; however, future investigations should continue to characterize more molecular mutations and their frequencies of occurrence in this select population. 406 Johnson Genetic risks of ICSI in male fertility Vol. 70, No. 3, September 1998