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1 The genetic origin of Klinefelter syndrome and its effect on spermatogenesis Merel Maiburg, M.D., a Sjoerd Repping, Ph.D., b and Jacques Giltay, M.D., Ph.D. a a Department of Medical Genetics, University Medical Center Utrecht, Utrecht; and b Academic Medical Center, Center for Reproductive Medicine, University of Amsterdam, Amsterdam, the Netherlands Klinefelter syndrome is the most prevalent chromosome abnormality and genetic cause of azoospermia in males. The availability of assisted reproductive technology (ART) has allowed men with Klinefelter syndrome to father their own genetic offspring. When providing ART to men with Klinefelter syndrome, it is important to be able to counsel them properly on both the chance of finding sperm and the potential effects on their offspring. The aim of this review is twofold: [1] to describe the genetic etiology of Klinefelter syndrome and [2] to describe how spermatogenesis occurs in men with Klinefelter syndrome and the consequences this has for children born from men with Klinefelter syndrome. (Fertil Steril Ò 2012;98: Ó2012 by American Society for Reproductive Medicine.) Key Words: Klinefelter syndrome, spermatogenesis, meiosis, sperm aneuploidy, TESE Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/maiburgm-genetic-origin-klinefelter-syndrome-spermatogenesis/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. The phenotype of what was later named Klinefelter syndrome was first described in 1942 by Harry Klinefelter (1). He reported nine men with gynecomastia, small testes, and azoospermia. In 1959, it was first demonstrated that men with Klinefelter syndrome have an additional X chromosome, resulting in a 47,XXY karyotype (2). Nowadays, it is known that a 47,XXY karyotype is found in 80% 90% of men with Klinefelter syndrome, whereas the remaining cases show a mosaic karyotype (46,XY/ 47,XXY), additional X chromosomes (e.g., 48,XXXYor 48,XXYY), or structurally abnormal X chromosomes (e.g., 47,X,iXq,Y) (3, 4). Klinefelter syndrome is the most prevalent chromosomal disorder in humans, with an estimated frequency of 1:500 to 1:1,000 men (3). It is also the most frequent genetic cause of azoospermia (5, 6). Most men with Klinefelter syndrome are diagnosed when they have failed to achieve a pregnancy and are diagnosed with azoospermia. However, a significant proportion of men with Klinefelter syndrome remain undiagnosed, probably because of the wide phenotypic variability and lack of knowledge of the syndrome among health professionals (3, 7). Since the introduction of intracytoplasmic sperm injection (ICSI) (8) and testicular sperm extraction (TESE) (9), a considerable number of men with Klinefelter syndrome have been able to father genetically own offspring. In light of this possibility for paternity, a review of the genetic etiology of the syndrome as well as its effects on spermatogenesis is useful in order to allow discussion of the potential risks of Received April 2, 2012; revised June 10, 2012; accepted June 12, 2012; published online June 29, M.M. has nothing to disclose. S.R. has filed a patent for markers of alterations in the Y chromosome and uses thereof (U.S. Patent Application ). J.G. has nothing to disclose. Reprint requests: Merel Maiburg, M.D., Department of Medical Genetics, University Medical Center Utrecht, Lundlaan AE Utrecht, the Netherlands ( m.maiburg@umcutrecht.nl). Fertility and Sterility Vol. 98, No. 2, August /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. this treatment. This review has two main objectives. First, we describe the genetic etiology of Klinefelter syndrome. In this section we address normal meiosis and focus on paternal and maternal causes of nondisjunction. Second, we describe how spermatogenesis occurs in men with Klinefelter syndrome and discuss the possible consequences for offspring from men with Klinefelter syndrome. GENETIC ORIGIN OF KLINEFELTER SYNDROME Normal Meiosis Before the first meiotic division, the amount of DNA is doubled (replication), resulting in 46 chromosomes, each consisting of two chromatids (2n,4c). The first meiotic division (reduction division) involves segregation of homologous chromosomes (2n,4c) and gives rise to haploid (23 chromosomes; 1n,2c) germ cells: two secondary spermatocytes (male meiosis) or one secondary oocyte and one polar body (female meiosis). In male meiosis, the second meiotic division (segregation of sister chromatids; 2c / c) gives VOL. 98 NO. 2 / AUGUST

2 VIEWS AND REVIEWS rise to spermatids that subsequently mature into spermatozoa. In female meiosis, the second meiotic division is finished only after fertilization, giving rise to a mature (fertilized) oocyte and a second polar body. Thus, each male germ cell entering meiosis eventually gives rise to four spermatozoa, whereas one complete round of female meiosis eventually produces one mature oocyte (10, 11). During prophase of meiosis I, homologous chromosomes pair and form connections called chiasmata. In male meiosis, the (largely nonhomologous) X and Y chromosome pair at the tips of their short and long arms, the pseudoautosomal regions 1 and 2 (PAR1 and PAR2). Paired homologous chromosomes exchange random DNA segments at the chiasmata, a process called crossing over, which results in a recombination of these segments. Crossing over takes place during prophase of meiosis I and is nonrandomly distributed along the chromosomes, with at least one exchange per chromosome arm (except for the short arms of acrocentric chromosomes). Genomewide recombination rates in female meioses are approximately 1.6- to 1.7-fold greater than in male meioses (12). The PAR1 region (2.6 Mb) contains one obligatory crossover (13). Pairing and crossing over at the smaller (320-kb) pseudoautosomal region (PAR2) at the tip of the long arms of the X and Y chromosomes is not essential for completing meiosis (14, 15). The purpose of crossing over is twofold: [1] to generate diversity within a population and [2] to ensure accurate segregation of chromosomes during meiosis I (16). The latter will be discussed below. Mechanisms Leading to Aneuploidy Nondisjunction is the failure of chromosomes to separate (disjoin) at anaphase during meiosis I (paired homologs), meiosis II (sister chromatids), or mitosis (sister chromatids) (11), giving rise to daughter cells with an aberrant number of chromosomes. The proper formation and resolution of chiasmata is necessary to keep the homologs in the right position during meiosis for their accurate separation into their daughter cells. Classical nondisjunction in meiosis I can result from failure to resolve chiasmata ( true nondisjunction ), premature resolution of chiasmata or failure to establish chiasmata ( achiasmate nondisjunction ). Another mechanism, premature separation of sister chromatids, can result in one complete chromosome and a single chromatid segregating together in meiosis I (17). Nondisjunction has long been regarded as the main mechanism leading to aneuploidy. Interestingly, however, using array comparative genomic hybridization (array-cgh) on first polar bodies, it has recently been shown that chromatid errors (premature separation; 3:2 ratio of sample vs. control DNA) were 11.5 times more common than whole chromosome errors (nondisjunction; 2:1 ratio) (18). Handyside et al. (19), who studied both polar bodies and the corresponding zygote by array-cgh, also conclude that almost all meiosis I errors are caused by premature division of sister chromatids. Finally, anaphase lagging is the failure of a chromosome or chromatid to be incorporated into a daughter cell following cell division (11). Chromosomes or chromatids not entering a daughter cell are lost, resulting in aneuploidy (monosomy) for that chromosome. Origin of XXY Aneuploidy It has always been assumed that most human trisomies originate from nondisjunction at maternal meiosis I (20). Indeed, paternal meiotic errors account for only 10% of autosomal trisomies. However, this is very different for sex chromosomal aneuploidies, including Klinefelter syndrome that results from a nondisjunction event in the father in nearly half of the cases (20, 21). In cases of Klinefelter syndrome with an additional maternal X, nondisjunction in either the first or second meiotic division is most likely to have occurred. In paternal cases, the additional X chromosome can only be the result of nondisjunction in the first meiotic division, because a meiosis II error will result in either XX or YY gametes (and therefore XXX or XYY zygotes) (20). As mentioned previously, premature separation of sister chromatids in meiosis I might be a more common cause of aneuploidy than originally thought (18). This mechanism could also underlie the origin of nonmosaic XXY cases of either paternal or maternal origin. Aberrant meiotic recombination has been shown to play an important role in the etiology of nondisjunction in Klinefelter syndrome (20, 22). The vast majority of Klinefelter cases of paternal origin result from a nullitransitional meiosis I nondisjunction, that is, a meiotic division with complete absence of recombination of the PAR regions, but transitional (with occurrence of crossing over) paternally derived cases also occur. Maternal cases with either absent or normal recombination have also been described (20, 23). In addition to studies using DNA markers in Klinefelter men (or fetuses) and their parents to assess recombination, direct analysis of 24,XY disomic sperm of a normal 46,XY male showed a significantly lower recombination frequency compared with 23,X or 23,Y sperm (24). Not only the number of crossing overs but also the localization of chiasmata seems to be important for meiosis to occur accurately. Crossing overs occurring too near or too far from the centromere have been described in autosomal trisomies: for example, trisomy 21 and trisomy 16. However, the position of recombination was normal in cases with Klinefelter syndrome as reviewed by Lamb et al. (25). Maternal age is a well-known risk factor for meiotic nondisjunction, especially in Down syndrome. Bojesen et al. found a 4-fold increase in the prevalence of Klinefelter cases with maternal age greater than 40 years compared with those with maternal age below 24 years (3). A maternal age effect was also shown in Klinefelter cases with a normal recombination pattern and in cases with postzygotic mitotic nondisjunction (resulting from a mitotic error early in the developing zygote) (20). The latter could be explained by the fact that in humans the first three mitotic divisions are solely controlled by maternal protein and RNA (26); with increasing maternal age, the chance of mitotic errors in the first cell divisions increases and therefore perhaps also the chance of Klinefelter syndrome of postzygotic origin. In a recent review of both epidemiologic studies and direct fluorescence in situ hybridization (FISH) studies, Fonseka et al. conclude that there is very little or no evidence for a correlation between paternal age and autosomal aneuploidy and some albeit debatable evidence for a relation with sex chromosomal trisomies and paternal age (27). 254 VOL. 98 NO. 2 / AUGUST 2012

3 Fertility and Sterility So far, aberrant recombination and increased maternal age are the only known factors associated with increased occurrence of nondisjunction. Theoretically, deletions within the PAR1 region could also hamper pairing of the X and Y chromosome and therefore impede recombination, increasing the risk of nondisjunction. This led to the hypothesis that interstitial Y chromosome deletions could be more common in paternally derived Klinefelter cases. However, a study of PAR1 deletions in Klinefelter men of paternal origin with demonstrated failure of recombination did not show an association between the presence of deletions and absence of recombination (28). Whether PAR2 deletions are more common in (paternally derived) Klinefelter men is unknown, but not expected given the fact that pairing at the PAR2 region is not required to complete meiosis (14, 15). The presence of other deletions on the long arm of the Y chromosome, such as deletions of AZF (azoospermia factor) regions in men with Klinefelter syndrome, has also been the subject of discussion. Although initial studies suggested an increased prevalence of AZF deletions (29), larger, more recent studies have not detected complete deletions of AZFa, AZFb, or AZFc in men with Klinefelter syndrome, and a prevalence of partial deletions (gr/gr and b2/b3 deletions) similar to karyotypically normal infertile men (30, 31). Ten percent to 20% of men with Klinefelter syndrome show a mosaic (mainly 46,XY/47,XXY) karyotype (4). Mosaic karyotypes arise from either nondisjunction in an early mitotic division of the developing (46,XY) zygote, or from loss of one of the X chromosomes of a 47,XXY conception ( trisomy rescue ) due to anaphase lagging. Genotype-phenotype Correlation Klinefelter syndrome shows a wide phenotypic spectrum, ranging from mild or aspecific (undetected) cases, to sterility, to learning or behavioral problems and/or physical features becoming manifest at an early age. Genomic imprinting, that is, the differential expression of genes depending on parental origin, has been proposed as an explanation for the clinical variability of several features like motor impairment, language problems and growth parameters (21). The variable CAG repeat length of the androgen receptor (AR) gene, which is inversely associated with androgen action, has also been studied to demonstrate a possible genotype-phenotype correlation in men with Klinefelter syndrome (32, 33). A longer AR CAG repeat has been associated with smaller testes (33), later onset, and slower progression of puberty and slower testicular degeneration (34) in Klinefelter syndrome. Whether genetic imprinting and/or variations in the AR gene CAG repeat length play a role in the fertility phenotype of Klinefelter syndrome, for example, the variable presence of spermatozoa in testicular tissue, is currently unknown. Role of X-chromosomal Fertility Genes The X chromosome contains over a thousand genes, 10% of which are specifically expressed in the testis (35). Mutation analysis of known (X-linked) fertility genes in infertile, karyotypically normal, patients has been largely unsuccessful (36), probably because the mutation rate in any of these genes is very small and/or because polymorphisms in these genes only have a small effect size and might only lead to fertility problems if associated with other genetic and/or environmental factors. However, a gene dosage effect rather than (inactivating) point mutations can be expected in Klinefelter syndrome. Even though X-inactivation occurs in Klinefelter men as in (46,XX) women (37), about 15% of X-chromosomal genes, as well as genes in the pseudoautosomal regions, consistently escape X-inactivation. An additional 10% is randomly inactivated (38). As a consequence, X-chromosomal genes escaping inactivation have a higher expression level in men with Klinefelter syndrome compared with karyotypically normal men. The overdosage of gene products may compromise testicular function or influence the meiotic process itself and therefore play a role in the etiology of infertility in Klinefelter males. SPERMATOGENESIS IN MEN WITH KLINEFELTER SYNDROME Infertility? One of the hallmark features of Klinefelter syndrome is azoospermia. From the first description of the syndrome in 1942, patients have therefore been regarded as infertile. However, sperm has been found in the ejaculate in 7.7% 8.4% of (apparently) nonmosaic Klinefelter patients (4, 39). Even a few spontaneous pregnancies in couples with a man with Klinefelter syndrome have been reported in the literature (40 43). Moreover, with currently available assisted reproduction techniques such as TESE, sperm can be recovered from the testes of Klinefelter patients in about half of the cases (44, 45). The rate of sperm retrieval might be higher using the more recent surgical technique of microdissection TESE (55% compared with 42% as reviewed by Fullerton et al.) (45). Using this sperm for ICSI offers a considerable number of Klinefelter men the opportunity to father their own genetic children. Together with these successes, concerns about the risk of (sex chromosomal) aneuploidy in these children have been raised. However, even though the aneuploidy rate of sperm from Klinefelter men appears to be increased compared with fertile controls (46), the risks for the offspring appear to be small, with 1 XXY pregnancy in more than 100 children born after TESE- ICSI in males with nonmosaic Klinefelter syndrome reported in the literature (7). How can this low (sex chromosomal) aneuploidy rate in children born from Klinefelter fathers be explained? This question has brought renewed attention to the discussion of how spermatogenesis (in particular meiosis) in men with Klinefelter syndrome occurs. Hypotheses Two hypotheses on how spermatogenesis occurs in Klinefelter men have been brought forward. According to some investigators, 47,XXY spermatogonia have the potential to complete meiosis, explaining both the increase in sex chromosomal aneuploidy rate as well as the presence of normal (haploid) spermatozoa (Fig. 1A) (47 50). Intuitively, this would be the expected mechanism because the vast majority of Klinefelter VOL. 98 NO. 2 / AUGUST

4 VIEWS AND REVIEWS FIGURE 1 Hypotheses on spermatogenesis in Klinefelter syndrome. (A) Meiosis of XXY cells. (B) Meiotic errors in 46,XY germ cells (testicular environment hypothesis). Maiburg. 47,XXY: origin and effect on spermatogenesis. Fertil Steril men are nonmosaic based on lymphocyte karyotypes and FISH results. According to others, spermatozoa of Klinefelter men arise from patches of 46,XY spermatogonial stem cells in the testes and the observed increase in aneuploid sperm compared with fertile controls is due to meiotic errors caused by a compromised testicular environment ( testicular environment hypothesis ; Fig. 1B) (51 54). Experimental Data One could think of several ways to address this question. Ideally, one would visualize the separation of sex chromosomes during meiosis in real time. Unfortunately, the development of a robust in vitro differentiation system of human spermatogonial stem cells has not been possible to date. However, investigators have looked at progenitor germ cells and the meiotic process itself in vivo, for example using FISH on pachytene figures (the meiotic stage in which crossing over occurs) (Table 1) (47, 48, 50 53, 55, 56). These investigators have reached different conclusions. Blanco et al., Bergere et al., and Sciurano et al. all found a 46,XY karyotype in approximately 100% of meiotic cells and concluded that XXY cells are meiotically incompetent (51 53). On the other hand, Foresta et al. (47), Yamamoto et al. (48), and Gonsalves et al. (53, 56) found an XXY karyotype in about half (56) to 100% (47) of spermatogonia and primary spermatocytes of Klinefelter men. The conclusion of both Yamamoto et al. and Foresta et al. that 47,XXY germ cells are able to complete spermatogenesis is questioned by Egozcue et al. (57) and Sciurano et al. (53), based on different arguments, including the absence of 46,XY spermatogonia in patients negative for spermatids and spermatozoa (48), technical shortcomings (48, 56), andsmall quantity of germ cells studied (47). Other investigators (49, 58 64) have used a different, indirect, approach and have deduced the most likely hypothesis from FISH results on spermatozoa (i.e., the end product of the meiotic process) as explained below, whereas others have combined the two approaches (47, 48, 51, 52). If the first hypothesis (segregation of XXY germ 256 VOL. 98 NO. 2 / AUGUST 2012

5 VOL. 98 NO. 2 / AUGUST TABLE 1 Spermatogenesis and sperm chromosome analysis in (apparently) nonmosaic Klinefelter patients. Study 47,XXY patients with spermatozoa (n) (Pre)meiotic cells n Normal X % (n) b Y % (n) Postmeiotic cells a XY % (n) Hyperhaploid Guttenbach 1997 c 1 (ejaculated) Not studied 2, (958) 48.8 (1,077) 1.4 (30) 1.2 (27) 0.1 (2) Estop (ejaculated) Not studied (5) 29.2 (7) 25.0 (6) 0 0 Foresta 1998 c 2 (ejaculated) Not studied 10, , Foresta (biopsies) Spermatogonia (n ¼ 10) Primary spermatocytes (n ¼ 20) XXY (100%) XX % (n) (25) 20.4 (11) 20.4 (11) 7.4 (4) (19) 19.0 (8) 23.8 (10) 7.1 (3) Levron (biopsies) Not studied (11) 33.3 (7) (9) 52.4 (11) (1) (10) 60.7 (17) (1) (8) 50.0 (10) 5.0 (1) 5.0 (1) (12) 45.5 (10) Morel 2003 c 1 (ejaculated) Not studied 5, Rives 2000 c 1 (ejaculated) Not studied 10, (5,024) 48.3 (4,895) 0.5 (55) 0.5 (46) 0.4 (37) Blanco (biopsy) Premeiotic cells (n ¼ 910) XXY (88.5%) XY (11.5%) Pachytene figures (n ¼ 36) XY (94.7%) Bergere (biopsies) Pachytene figures (n ¼ 20) XY (100%) (49) 40.8 a (49) 18.3 a (22) YY % (n) (11) 29.4 (5) 5.9 (1) (11) 56.6 (17) (12) 48.1 (13) (1) 2,400 (total) 52.0 (1,247) 41.6 (998) 2.8 (68) 1.0 (25) 0.4 (9) Yamamoto (biopsies) Spermatogonia (n ¼?) XXY > XY Ferlin 2005 c 7 (ejaculated) Not studied NR Gonsalves (biopsy) Meiotic cells (n ¼ 100) XXY (47%) XY (53%) Sciurano (biopsies) Pachytene spermatocytes (n ¼ 92) XY (100%) a For those studies distinguishing secondary spermatocytes, spermatids, and/or spermatozoa, these numbers were added up. b Absolute numbers are not reported in all studies and therefore not reported in this table for all cases. c Reviewed by Ferlin et al Maiburg. 47,XXY: origin and effect on spermatogenesis. Fertil Steril Not studied Not studied Fertility and Sterility

6 VIEWS AND REVIEWS cells) is true, then, theoretically, one would expect an increase in both XY and XX disomy in spermatozoa studied (with a 2:1 ratio if segregation occurs randomly), but no increase in YY disomy or nullisomy (Fig. 1A). The second hypothesis (testicular environment hypothesis) implies meiotic errors in XY germ cells to explain the increased sperm aneuploidy rate. This will result in an increase in XY spermatozoa (XY disomy) and spermatozoa nullisomic for the sex chromosomes if nondisjunction occurs in the first meiotic division. Nondisjunction in meiosis II will result in an increase in XX disomy, YY disomy, and sex chromosomal nullisomy (Fig. 1B). Moreover, if karyotypically normal germ cells are prone to meiotic errors because of the compromised testicular environment, one would also expect an increase in disomy and nullisomy for all other chromosomes (autosomes) as well as diploidy (spermatozoa with 46 chromosomes), which cannot be explained by meiosis of XXY cells. Another difference is the expected ratio of X:Y bearing euploid spermatozoa, which is 1:1 in case of meiosis of XY cells but will show an excess of X-bearing euploid spermatozoa with segregation of XXY cells (54). Results of all these studies that have assessed the chromosomal status of spermatozoa of men with Klinefelter syndrome are summarized in Table 1. Nullisomy of the sex chromosomes is not always scored, because of difficulties in interpretation of an absent signal (true absence of a sex chromosome or failure of hybridization) (63) therefore is not included in the table. Some of these studies indeed show an excess of X-bearing spermatozoa related to Y-bearing spermatozoa (49, 60), yet in other studies the proportion was 1:1 (51, 52, 63). Studies in favor of the testicular environment theory (i.e., spermatozoa are derived from 46,XY precursors and the increased aneuploidy rate is considered to be due to the altered endocrine environment of the testis) indeed show an increase of YY disomy (52, 61, 63, 64), that would not be expected in case of meiosis of 47,XXY cells as described above. Even though the total aneuploidy rates in the abovementioned studies are increased, percentages are in general much lower than the (theoretically) expected sex chromosomal aneuploidy rate of up to 50% in case of 47,XXY meiosis. Moreover, studies on aneuploidy rates in men with any type of infertility (including men with a normal karyotype) showed a 2 10 times increased frequency of sperm aneuploidy compared with fertile controls (65). Most of the above-mentioned studies used FISH to detect the number of sex chromosomes, and none of them show the complete karyotype of single cells. As a consequence, autosomal aneuploidies will largely remain undetected, which might bias the results in favor of the 47,XXY meiosis hypothesis. Those studies mentioning the sex chromosomal content of Sertoli cells show an XXY karyotype in all (47, 48) or the majority (53) of these cells. This finding suggests that if the testicular environment (XY) theory is correct, the XY cells are confined to progenitor germ cells and do not necessarily result from somatic mosaicism. In summary, studies have shown both 46,XY and 47,XXY progenitor cells. Total sperm aneuploidy rates in men with Klinefelter syndrome, including increases of YY disomy in some studies, are increased compared with karyotypically normal controls, although the ratios of different aneuploidies as well as the ratio of X:Y-bearing euploid spermatozoa differ between studies. Altogether, although several investigators state to have shown strong or definite evidence for either the 46,XY or the 47,XXY progenitor cell hypothesis, it would seem that a definite conclusion cannot be drawn at this time. In our opinion, the strongest, direct evidence comes from those studies investigating pachytene figures, because they directly show meiotic cells. These studies are in favor of the testicular environment hypothesis, showing an XY pattern in nearly all cells (51 53), with the exception of Gonsalves et al., who found an XY pattern in only 53% of meiotic cells (56). Among those direct studies supporting the XXY hypothesis, most study premeiotic cells (spermatogonia) rather than truly meiotic cells (pachytene spermatocytes). Those studies using FISH on spermatozoa show only indirect evidence, and report variable results and conclusions. Although it cannot be excluded that the two hypotheses may actually coexist (49), we feel that based on all available data there are stronger arguments to support the testicular environment hypothesis, assuming that sperm in men with Klinefelter syndrome are produced by a subset of 46,XY spermatogonia in the testes. Animal Model What can be learned from animal studies? The testicular environment hypothesis is supported by the findings of Mroz et al. (66), who showed that none of the 73 germ cells from XXY male mice that were studied had two X chromosomes. In another study by the same investigator, spermatids from eight adult XXY mice and five control mice were investigated (54). They found a threefold increase of XX and YY disomy (expected to result from a meiosis II error of 46,XY cells; see above) and a fourfold increase of diploid spermatozoa compared with control mice. Moreover, they found no evidence for an increase in X-bearing spermatozoa among euploid spermatozoa, as would be expected if spermatozoa had been derived from XXY progenitor cells. This led to their conclusion that the increase in sperm chromosome abnormalities is attributable to malsegregation in XY germ cells. Burgoyne et al. showed that errors in synapsis are accompanied by gene inactivation and an impairment of meiosis (67). Conversely, inadequate silencing of sex chromosomal genes in XYY mice resulted in meiotic arrest (68). A similar mechanism might explain the inability of XXY cells to undergo meiosis. Origin of 46,XY Germ Cells in Apparently Nonmosaic Klinefelter Men Following this discussion, another question could be asked: if germ cells are indeed 46,XY, then how would these cells arise? Is the loss of one X chromosome a random event, or due to specific characteristics of the X chromosome(s), for example X-inactivation, as suggested by Sciurano et al. (53)? Levron 258 VOL. 98 NO. 2 / AUGUST 2012

7 Fertility and Sterility et al. speculated that during multiplication of the primordial germ cells in the prenatal testis correcting mitotic errors might give rise to isolated testicular mosaicism; normal germ lines survive to produce sperm later during adulthood (61). Mroz et al. demonstrated in a mouse model that surviving germ cells are all XY and restricted to single continuous segments, indicating that they arose from clonal proliferation of single germ cells that had lost an X chromosome (66). Consequences for Children Born after TESE/ICSI A recent overview of more than 100 children born from men with Klinefelter syndrome showed only 1 child with an abnormal karyotype (XXY) (in the paper by Fullerton et al., the single case has been counted twice by mistake) (7). How does the absence of high aneuploidy rates in children born after TESE/ICSI in Klinefelter men correlate with the observed increased aneuploidy rate in the sperm of Klinefelter men? An explanation might be reduced implantation potency of karyotypically abnormal embryos (69). It is important to note that even though sperm aneuploidy rates are increased in men with Klinefelter syndrome compared with the normal population, they are comparable to the rates observed in karyotypically normal ICSI candidates with oligoasthenoteratozoospermia (46). CONCLUSION We described the genetic origin of Klinefelter syndrome and its effect on spermatogenesis. Men with Klinefelter syndrome have an extra paternal X chromosome in nearly half of all cases. In nonmosaic Klinefelter syndrome of both paternal and maternal origin, the extra X chromosome is the result of meiotic nondisjunction or possibly, as recently described, of premature separation of sister chromatids. So far, an increased maternal age and aberrant recombination are the only known factors to be associated with the occurrence of nondisjunction. With regard to spermatogenesis in men with Klinefelter syndrome, the strongest evidence is in support of the testicular environment hypothesis, implying that spermatozoa of Klinefelter men arise from patches of 46,XY spermatogonial stem cells in the testes and that the observed increase in aneuploid sperm compared with fertile controls is due to meiotic errors caused by a compromised testicular environment. Despite this increased aneuploidy rate, children born from men with Klinefelter syndrome do not appear to be at increased risk for aneuploidies. REFERENCES 1. Klinefelter HF, Reifenstein EC, Albright F. Syndrome characterised by gynecomastia, aspermatogenesis without a-leydigism, and increased excretion of follicle-stimulating hormone. J Clin Endocrinol 1942;2: Jacobs PA, Strong JA. A case of human intersexuality having a possible XXY sex-determining mechanism. Nature 1959;183: Bojesen A, Juul S, Gravholt CH. 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