Meiotic chromosome abnormalities in fertile men: are they increasing?

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1 Meiotic chromosome abnormalities in fertile men: are they increasing? Laia Uroz, Ph.D., a Osvaldo Rajmil, Ph.D., b and Cristina Templado, Ph.D. a a Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autonoma de Barcelona, Bellaterra; and b Servei d Andrologia, Fundacio Puigvert, Barcelona, Spain Objective: To determine the basal frequencies of meiotic chromosome abnormalities in fertile men. Design: Descriptive design. Setting: Research university laboratory and clinical andrology service. Patient(s): Seventeen fertile donors undergoing vasectomy. Intervention(s): Analysis of testicular biopsies. Main Outcome Measure(s): Meiotic chromosome abnormalities in metaphase I spermatocytes. Result(s): A total of 1,407 spermatocytes I was analyzed. The main meiotic abnormality was absence or low chiasma number of individual bivalents (23.4%), followed by structural (3.3%) and numerical (0.7%) abnormalities. Sex chromosomes and G-group chromosomes were the most commonly found as univalents at metaphase I. Statistically significant heterogeneity was found for meiotic abnormalities among fertile men, caused by interindividual variation in the level of dissociated sex chromosomes (ranging from 3.2% to 43.7%). The mean total percentage of meiotic abnormalities in spermatocytes I from fertile men was 27.4%, 1.7 times higher than those described a few decades ago in fertile and even in infertile men. Conclusion(s): Fertile men are a heterogeneous group for meiotic errors, with individuals showing percentages of meiotic abnormalities as high as 50%. From these findings, caution is recommended when using meiotic studies to diagnose and provide genetic counselling to patients consulting for infertility. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Meiotic abnormalities, fertile men, spermatocytes, achiasmate bivalents Chromosome abnormalities are responsible for approximately 20% of male infertility (1), including somatic chromosome abnormalities and meiotic chromosome disorders present in germ cells of men with a normal karyotype. Meiotic chromosome disorders may cause partial or total meiotic arrest, resulting in oligozoospermia and azoospermia, respectively. Moreover, these abnormalities could lead to chromosomally abnormal spermatozoa, causing pregnancy loss and affected offspring. Meiotic studies allow the identification of meiotic abnormalities, and their use is especially relevant when diagnosing patients with idiopathic infertility. As a consequence of the increased frequency of de novo chromosome abnormalities found in pregnancies obtained by intracytoplasmic sperm injection (2), meiotic studies have been recommended in infertile men undergoing this technique (3,4). Despite their significance, direct meiotic studies are scarce, because they are carried out in spermatocytes obtained from a testicular biopsy. Most meiotic studies have applied conventional cytogenetic techniques to metaphase I (MI) spermatocytes from heterogeneous infertile series to determine the causes of their infertility (5 9). Received March 9, 2010; revised June 11, 2010; accepted June 16, 2010; published online August 2, L.U. has nothing to disclose. O.R. has nothing to disclose. C.T. has nothing to disclose. This research was supported by the Ministerio de Ciencia y Tecnologıa, Spain (BFI ) and the Generalitat de Catalunya, Spain (2005FI00399, 2009SGR-01107). Reprint requests: Cristina Templado, Ph.D., Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autonoma de Barcelona, Bellaterra, Barcelona 08193, Spain (FAX: ; cristina.templado@uab.es). These studies revealed that meiotic arrest involved 18.5% of infertile men, and meiotic chromosome abnormalities affected 6% of cases (reviewed by Egozcue et al. [8]). In selected patients with severe oligoasthenozoospermia the incidence of meiotic chromosome disorders may increase to 17.5% (3). The most frequent meiotic chromosome disorder described in infertile men is abnormal chiasma frequency, observed for the first time as a decrease in the number of chiasmata affecting all bivalents in some or all cells (6, 10 17). Templado et al. (18) reported that this reduction in the number of chiasmata can affect only individual bivalents. A low number of chiasmata in MI results in monochiasmate medium-sized bivalents and in dissociated small univalents. The presence of two dissociated sex chromosomes has been described in all series of fertile and infertile men, and it has been suggested that percentages higher than 30% may lead to infertility (19). To our knowledge, cytogenetic meiotic analyses in MI spermatocytes from fertile men are scarce, and little is known about the baseline chromosomal abnormalities in these individuals. Several of these studies have been carried out in infertility clinic patients with normal meiosis (5, 9), and all of them reported the dissociation of sex chromosomes (5, 7, 9). One single series of eight fertile men (6) described dissociation of both sex chromosomes and small autosomes. We carried out meiotic chromosome studies, using the airdrying technique, in MI spermatocytes from 17 fertile male donors undergoing surgical vasectomy. The main purposes of this work were to investigate [1] the basal frequencies of meiotic chromosome abnormalities in fertile men, and [2] the differences between our results and those previously reported by other authors in similar studies /$36.00 Fertility and Sterility â Vol. 95, No. 1, January doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 MATERIALS AND METHODS Subjects Testicular samples were collected from 17 healthy fertile men, selected from individuals attending the Andrology Service of Puigvert Foundation for vasectomy. The ages of the donors ranged from 28 to 47 years (mean, 38.3 years). All of them had fathered at least one naturally conceived child. Among these 17 donors, 7 were smokers (cases 1, 3, 4, 10, 14, 15, and 16), and none had been exposed to known mutagens or radiation. The frequencies of meiotic chromosome abnormalities in spermatocytes I and II from cases 1 and 2 have been described elsewhere (20) using the centromere-specific multicolor fluorescence in situ hybridization (FISH) technique, and these data have not been included in this work. The study was approved by our University and institutional ethics committees, and all donors signed an informed consent form before surgery. MI Spermatocyte Preparation Testicular tissue was collected under local anaesthesia during surgical vasectomy, through the same scrotal incision used for deferens resection. Meiotic cells were fixed using the air-drying technique (21) with minor modifications (22). Meiotic chromosome preparations were preserved at 20 C until 4 0,6-diamidino-2-phenylindole (DAPI) stain. DAPI Stain Slides were washed in 1 phosphate-buffered saline for 5 minutes, treated with 0.4 standard saline citrate/0.3% NP-40 for 2 minutes at 71 C and with 2 standard saline citrate/0.1% NP-40 for 5 minutes, and dehydrated in an ethanol series. Spermatogenic cells were stained with DAPI in antifade solution (DAPI II; Vysis, Downers Grove, IL). Data Collection and Analysis Spermatocyte images were captured under an epifluorescence microscope (Olympus Bx60, Hamburg, Germany) and using a Power Macintosh G4 (Apple, Cupertino, CA). The DAPI-stained metaphases I were analyzed using SmartCapture software (Digital Scientific, Cambridge, United Kingdom). Metaphase II spermatocytes were not analyzable owing to the high number of overlapping chromosomes and their characteristic chromatin spiralization. Metaphase analysis was performed by two different observers, and chromosome abnormalities were classified according to the International System for Human Cytogenetic Nomenclature (23). To avoid technical artefacts, a conservative estimate of aneuploidy was calculated as being twice the hyperploidy frequency. Hypoploid metaphases were not included in this study, except those showing other chromosome abnormalities. Chiasma frequency was counted in each individual in 15 well-spread spermatocytes I with a normal karyotype (23,XY). Statistical Analyses To evaluate the interdonor variation in the incidence of meiotic chromosome abnormalities, the two-tailed Fisher exact test was performed. To analyze correlation between age and abnormality frequencies in the 17 fertile donors, the Spearman correlation test was applied. To investigate whether there were statistically significant differences among individuals in terms of chiasma frequency, we used the Kruskal-Wallis nonparametric test. RESULTS A total of 1,407 spermatocyte metaphases I was analyzed using the DAPI stain technique (Fig. 1). The DAPI-stain images are similarly informative to those reported in classic meiotic studies using uniform stain. In our laboratory, DAPI stain is used because the same spreads may be sequentially subjected to FISH techniques for further studies. The percentages of meiotic chromosome abnormalities are shown in Table 1, and abnormal karyotypes are listed in Table 2. Meiotic chromosome abnormalities (27.4%) affected principally the number of chiasmata of individual bivalents (85%), followed by structural (12%) and numerical (3%) abnormalities. Low chiasma count was observed both as [1] two separated univalents for sex chromosomes (22.1%) (Fig. 1A) or for small autosomes (0.4%) (achiasmate chromosomes) (Fig. 1B), and [2] medium-sized lineal bivalents with a single distal chiasma (0.9%) (monochiasmate bivalents). Structural abnormalities (3.3%) were principally acentric fragments (Fig. 1C), followed by chromosome breaks (Table 2). Trisomy (0.4%), found in most cases as a bivalent and an extra univalent, was significantly correlated with age (P¼.005). Significant interdonor heterogeneity was observed for the total meiotic chromosome abnormalities (range, 8.1% 46.5%) (P<.001) FIGURE 1 Metaphase I spermatocytes showing meiotic abnormalities. (A) Achiasmate sex bivalent, observed as two separated univalents. (B) Achiasmate autosomal bivalent with the size of G-group. A diagram of this abnormality is shown in the upper right corner. (C) Presence of an acentric fragment. Stain with DAPI allows identification of sex chromosomes and occasionally bivalents 1 and 9. I ¼ univalent. 142 Uroz et al. Meiotic abnormalities in fertile men Vol. 95, No. 1, January 2011

3 Fertility and Sterility â 143 TABLE 1 Meiotic chromosome abnormalities and chiasma count in 17 fertile men. Donor Age (y) MI analyzed Metaphase I Achiasmate and monochiasmate II (%) Numerical abnormality (%) Sex Structural chromosome Autosome Trisomy Aneuploidy a abnormality (%) Total meiotic abnormality (%) Chiasma count, mean ± SD (18.4) 2 (1.6) 1 (0.8) 2 (1.6) 5 (4.0) 32 (25.6) (16.3) 3 (1.5) (3.9) 44 (21.7) (40.0) (40.0) (26.1) 1 (0.9) 1 (0.9) 2 (1.7) 2 (1.7) 35 (30.4) (26.7) 1 (0.6) (4.3) 51 (31.7) (3.2) 1 (1.6) (3.2) 5 (8.1) (26.1) (1.1) 25 (27.2) (8.9) (3.6) 7 (12.5) (24.5) (7.5) 17 (32.1) (20.0) (4.0) 6 (24.0) (25.4) (1.5) 18 (26.9) (43.7) 3 (4.2) (7.0) 39 (54.9) (26.3) 3 (3.2) (29.5) (11.8) (11.8) (17.1) 0 2 (2.9) 4 (5.7) 3 (4.3) 19 (27.1) (25.8) 1 (1.0) (3.1) 29 (29.9) (13.2) 2 (2.9) 1 (1.5) 2 (2.9) 3 (4.4) 16 (23.5) Total , (22.1) 17 (1.2) 5 (0.4) 10 (0.7) 47 (3.3) 385 (27.4) Note: Spermatocytes presenting more than one meiotic chromosome abnormality were counted in each category. II ¼ bivalents. a Conservative aneuploidy (2 hyperploidy).

4 TABLE 2 Abnormal karyotypes detected in 1,407 spermatocytes I from 17 fertile men. Meiotic abnormality n (%) Achiasmate and monochiasmate bivalents 328 (23.3) Sex chromosomes a 311 (22.1) 24,X,Y (297 cells) Autosomes 17 (1.2) 23,XY,II(B,B)(xma ¼ 1) 23,XY,II(C,C)(xma ¼ 1) (7 cells) 24,X,Y,II(C,C)(xma ¼ 1) 23,XY,II(C,C)(xma ¼ 1),þace b 25,X,Y,II(C,C)(xma ¼ 1),II(C,C) (xma ¼ 1),-II(D),þI(D),þI(D) 23,XY,-II(G),þI(G) (2 cells) 23,XY,-II(G),þI(G),þI(G) (2 cells) Numerical abnormalities 10 (0.7) Diploidy 0 Trisomy 5 (0.4) 25,X,Y,þI(C) 25,X,Y,þII(E) 24,XY,þI(E) (2 cells) 25,X,Y,þI(G) Conservative aneuploidy 10 (0.7) (2 hyperploidy) Structural abnormalities 47 (3.3) 23,XY,þace (27 cells) 24,X,Y,þace (8 cells) 23,XY,þ2ace 23,XY,II(C,C)(xma ¼ 1),þace b 23,XY,chtb(X),þace 23,XY,chrb(1) 23,XY,2chrb(C) 24,X,Y,del(C)(q?) 24,XY,þmar 22,XY,III(DqGq) 23,XY,pvz(E) Total meiotic abnormalities MI 385 (27.4) Note: Hypoploid spermatocytes were not included. I ¼ univalent; II ¼ bivalent; III ¼ trivalent; xma ¼ chiasma count; ace ¼ acentric fragment; chtb ¼ chromatid break; chrb ¼ chromosome break; del ¼ deletion; mar ¼ marker chromosome. a Fourteen spermatocytes I with sex chromosome univalents also had other chromosome abnormalities. b One cell had both synaptic and structural abnormalities. and for dissociated sex chromosomes (range, 3.2% 23.5%) (P<.001). One individual (case 12) showed significantly elevated frequencies (P<.001) of meiotic chromosome abnormalities compared with the remaining 16 donors analyzed. Significant interindividual variation in the chiasma count was detected (P<.001), with a total mean (SD) number of chiasmata per cell of (Table 1). DISCUSSION The aim of this study was to describe the basal frequencies of chromosome abnormalities in MI spermatocytes from fertile men, including the presence of unpaired homologous chromosomes, monochiasmate bivalents, and numerical and structural abnormalities. Unexpectedly, we detected an increase in the frequencies of meiotic abnormalities with regard to those described in fertile and infertile men in similar studies of past decades. Meiotic Chromosome Abnormalities in Fertile Men The most frequent abnormality found in fertile men was the presence of individual homologues with low number or absence of chiasma, as previously described in series of infertile and sterile men (8, 24). However, our results are not easily comparable with those obtained in these studies, because they were focused on assessing the meiotic causes of the reproductive failure and reported only the frequencies of patients showing meiotic abnormalities. Chromosomes with absence of chiasma consisted essentially of univalents for sex chromosomes and G-group autosomes, as previously reported in MI spermatocytes of fertile male donors (6) and in pachytene studies in normal men (25). Moreover, chromosome 21 and sex chromosomes are the most commonly involved in disomies in spermatozoa from healthy men (reviewed by Templado et al. [26]) and in trisomies among aneuploid newborns (27). The mean percentage of unpaired homologous chromosomes is higher than that described in the other similar meiotic study (6). Theoretically, the absence or decreased number of chiasmata in individual bivalents may originate from one or more of the following disorders during the first meiotic division: [1] pairing or synaptic errors, [2] absence of recombination, and [3] precocious resolution of chiasmata. Technical approaches used in this study do not help to elucidate the mechanisms from which achiasmate homologues arise. G-group and sex chromosome bivalents usually have one single chiasma in meiosis I (9), because G-group chromosomes are the smallest in the karyotype, and sex chromosomes pairing is limited to the pseudoautosomal region. Thus, they are prone to appear as univalent chromosomes that could segregate at random during anaphase I, leading to meiotic nondisjunction in approximately 50% of cases and, in turn, to aneuploid germ cells. In addition, C-group bivalents with a single distal chiasma may also be prone to nondisjunction during meiosis I (28), because they may mono-orient toward the same spindle pole (29). Clinical consequences of these meiotic disturbances may be mild or moderate oligozoospermia and aneuploid conceptions. Structural abnormalities in spermatocytes I are principally nonrejoined breaks, as previously described in spermatozoa using the human hamster technique (30, 31). The percentage of structural abnormalities in spermatocytes I is lower than that described in spermatozoa from healthy males (6.6%) (reviewed by Templado et al. [26]), supporting the hypothesis that chromosome aberrations mainly arise during late spermatogenesis, a repair-deficient stage prone to DNA lesions (30). The positive correlation between the frequency of trisomy in spermatocytes I and age found in our study could be explained by an increase in the number of spermatogonial self-renewal divisions before entering meiosis, and consequently a higher probability of chromosome missegregation. Alternatively, this correlation could also be due to a diminished efficiency of the pachytene checkpoint (32, 33) with increasing age. According to our finding, FISH studies in spermatozoa from control males demonstrated increased frequencies of disomy for chromosomes 1 and 9 with age (34, 35). The effect of advancing age on trisomy in MI spermatocytes remains the subject of further studies specifically designed for this purpose. The mean percentage of meiotic abnormalities in MI spermatocytes (27.4%) is considerably higher than that described in spermatozoa from normal men (8.6%) (2% numerical abnormalities and 144 Uroz et al. Meiotic abnormalities in fertile men Vol. 95, No. 1, January 2011

5 TABLE 3 Percentages of meiotic chromosome abnormalities in spermatocytes I from series of fertile and infertile men. Fertile men Infertile men Meiotic abnormality Present work Skakkebaek et al. (6) Skakkebaek et al. (6) Achiasmate and monochiasmate II X,Y 22.1 a b Autosomal univalents Monochiasmate bivalents ND c Trisomy Structural abnormalities 3.3 a Note: II ¼ bivalents. a Significantly higher (P<.001) vs. other series of both fertile and infertile men. b Mean frequency of dissociated sex chromosomes in infertile men was calculated from data reported by Luciani (5), Skakkebaek et al. (6), Chandley et al. (7), and Laurie and Hulten (9). c Mean frequency not described, ranging from 0 to 100%, depending on the patient. 6.6% structural aberrations) (26), principally owing to the high levels of unpaired sex chromosomes (22.1%). Arrest of chromosomally abnormal spermatocytes monitored by meiotic checkpoints could explain the decrease in numerical abnormalities in spermatozoa (36, 37). Studies in mice support that the frequency of univalents in MI is overestimated, probably because of a delay in apoptotic elimination of the arrested cells (38). Failures of meiotic checkpoints could be responsible for the high frequencies of chromosome abnormalities in spermatozoa detected in some infertile men (reviewed by Handel et al. [39]). Frequencies of Meiotic Chromosome Abnormalities Vary Among Fertile Men Meiotic chromosomal abnormalities present a wide range among the fertile men studied because of interindividual differences in the frequency of dissociated sex chromosomes, which was surprisingly variable among individuals (from 3.2% to 43.7%). In agreement with our finding, a previous meiotic analysis of fertile individuals described a wide range of percentages for sex chromosome univalents (from 0 to 26%) (6), and studies in spermatozoa from fertile men also reported heterogeneity for disomy XY (reviewed elsewhere [26, 40]). This interdonor variation indicates that some fertile men consistently have high percentages of achiasmate homologues (e.g., donor 12) and may be at an increased risk of fathering aneuploid offspring. In fact, increased disomy levels have been reported in spermatozoa from men with Down (41), Turner (42), and Klinefelter syndrome children (43) for the chromosome pair implicated in their aneuploid offspring. Complete meiotic arrest, affecting 18.5% of infertile males (8), was not found in our fertile series. However, three of the donors (cases 3, 10, and 14) exhibited a low number of MI spermatocytes and, in general, scarce meiotic cells. A possible explanation for this decreased levels of germ cells is that hypospermatogenesis, which is highly prevalent in infertile men (60% of infertile patients) (44), may also be a common feature among fertile male donors. Meiotic studies have been indicated in males with idiopathic infertility to elucidate the causes of their fertility problems (2, 3). Both the interdonor heterogeneity and the high frequency of meiotic abnormalities found in some fertile men in this work suggest a cautious interpretation of meiotic studies when diagnosing and counselling infertile patients. Are Human Meiotic Abnormalities Increasing in the Male Population? The percentages of dissociated sex chromosomes and structural abnormalities found in our fertile series are higher than those found in fertile (6) and infertile men (5 7, 9) in the 1970s and 1980s (Table 3). This increase in the frequencies of meiotic abnormalities could be associated with differences in donor age among studies. Unfortunately, no definitive conclusion could be drawn about this relationship, because the mean age of our donors (38.3) was higher than that reported in one of the series (6) but similar to those described in the other two (5, 9). Nevertheless, the age range of our donors is similar to the current reproductive age, and thus our findings are generalizable to the fertile population. Although classic meiotic studies considered that percentages of sex chromosome univalents higher than 30% result in infertility (reviewed by Luciani and Stahl [19]), two of our fertile donors had values of 40% and 44% (cases 3 and 12, respectively). Recently, elevated levels of non-crossover sex chromosomes (up to 44%) have also been found in prophase I spermatocytes from men with normal spermatogenesis (25, 45). High frequencies of sex chromosome univalents may lead to 47,XXY and 45,X conceptuses. In accordance with our data, a recent study (46) described an increase of Klinefelter syndrome offspring of paternal origin compared with earlier newborn surveys, hypothesizing that some factor may be interfering with pairing and recombination during the paternal meiosis I. In fact, a direct study of mouse oocytes (47) observed that exposure to bisphenol A results in increased synaptic abnormalities and recombination levels. Moreover, several studies in spermatozoa associated high frequencies of diploidy and disomy with medical treatments, environmental exposure, and lifestyle habits (reviewed by Pacchierotti et al. [48]). These findings may indicate the existence of new factors that increase the frequencies of meiotic chromosome abnormalities and, in turn, may contribute to the unexplained decrease in both the mean sperm count (reviewed elsewhere [49, 50]) and fertility rates (reviewed by Joensen et al. [51]). In conclusion, fertile men are a heterogeneous group for the rate of achiasmate sex chromosomes. Moreover, they show increasing Fertility and Sterility â 145

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