Hum. Reprod. Advance Access published online on May 15, 2008
Human Reproduction, doi:10.1093/humrep/den027
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The relationship between meiotic recombination in human spermatocytes and aneuploidy in sperm
1 Department of Medical Genetics, University of Calgary, 3330 Hospital Dr, NW, Calgary, AB, Canada T2N 4N1 2 Department of Preventive Medicine, Northwestern University Medical School, Chicago, IL 60611-4402, USA 3 Department of Urology, University of California San Francisco, San Francisco, CA 94143-1695, USA 4 Department of Obstetrics and Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143-1695, USA 5 Present address: Hefei National Laboratory for Physical Sciences, Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China
6 Correspondence address. Tel: +1-403-220-7520; Fax: +1-403-210-7931; E-mail: rhmartin{at}ucalgary.ca
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Abstract |
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BACKGROUND: We have previously demonstrated that a decreased recombination frequency between human X and Y chromosomes is associated with the production of aneuploid 24,XY sperm. This study's aim was to determine the relationship between recombination frequency in human pachytene spermatocytes and aneuploidy frequencies in individual chromosomes in sperm from the same men.
METHODS: Six previously fertile vasectomy reversal patients donated testicular tissue for meiotic analysis of pachytene spermatocytes using immunocytogenetic techniques for visualization of the synaptonemal complex and recombination sites (MLH1). Individual meiotic chromosomes were identified with centromere-specific multicolor fluorescence in situ hybridization (FISH), and the number of MLH1 signals was recorded for individual chromosomes. An ejaculated sperm sample was obtained from each patient 2–26 months post-reversal for FISH analysis of sperm aneuploidy frequencies of chromosomes 1, 9, 13, 21, X and Y.
RESULTS: There was no significant correlation between meiotic recombination frequency and sperm aneuploidy for any individual chromosome. Similarly, there was no correlation between aneuploid sperm and bivalents with no recombination.
CONCLUSIONS: The study provides unique data on intra-individual human recombination and aneuploidy events. It also demonstrated for the first time that men do not have an increased frequency of sperm aneuploidy 5–9 years post-vasectomy.
Key words: meiotic recombination/human sperm aneuploidy/synaptonemal complex/pachytene spermatocytes/non-disjunction
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Introduction |
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Pairing and recombination of homologous chromosomes during meiotic prophase I are essential for chromosome segregation and the formation of haploid gametes. Improper chromosome segregation during meiosis, i.e. non-disjunction, results in genetically unbalanced oocytes or sperm. If these gametes participate in fertilization, the outcome is the formation of an aneuploid embryo—the leading known cause of pregnancy loss, mental impairment and developmental disabilities (Lamb and Hassold, 2004
).
The association between meiotic recombination and aneuploidy has been well-documented in model organisms (Koehler et al., 1996; Yuan et al., 2000; Molnar et al., 2001). In the past several years, a number of linkage analysis studies have demonstrated that this relationship is also important in humans. Absent or reduced levels of meiotic recombination or suboptimally positioned recombination events have been associated with non-disjunction in both males (Hassold et al., 1991; Lorda-Sanchez et al., 1992; Savage et al., 1998) and females (Sherman et al., 1991; Lamb et al., 1997). Because the sex chromosomal bivalent seems particularly susceptible to non-disjunction in human males, with the majority of sex chromosomal aneuploidies originating from paternal errors (Jacobs et al., 1990; MacDonald et al., 1994), we sought to determine if a reduced recombination frequency is associated with non-disjunction of the X and Y chromosomes in individual sperm. Initially, we performed single sperm PCR analysis on a normal 46,XY male to determine if there was any alteration in the recombination frequency of aneuploid 24,XY sperm compared with unisomic sperm (23,X or 23,Y) (Shi et al., 2001). The frequency of recombination between the two DNA markers was 38.3% for unisomic sperm, but there was a highly significant decrease in recombination in aneuploid sperm (25.3%). This research was the first direct evidence that altered recombination has an effect on non-disjunction in human gametes.
Studies of infertile men have also begun to address this issue. ICSI (Palermo et al., 1992) now allows severely infertile men the opportunity for biological fatherhood. However, it is also clear that there is genetic risk in using sperm from infertile men for ICSI. We (Moosani et al., 1995; McInnes et al., 1998) and others (Pang et al., 1999; Pfeffer et al., 1999; Vegetti et al., 2000) have demonstrated that even karyotypically normal infertile men have an increased frequency of aneuploid sperm and this is particularly marked for infertile men with non-obstructive azoospermia (Bernardini et al., 2000; Palermo et al., 2002; Martin et al., 2003a). We are interested in understanding whether this increased frequency of aneuploidy in infertile men is also associated with defects in meiotic recombination. Luckily, immunofluorescence methods that directly visualize important meiotic proteins now make this research possible (Barlow and Hulten, 1998; Anderson et al., 1999; Hassold et al., 2000; Sun et al., 2004a). Antibodies against synaptonemal complex (SC) proteins SCP1 and SCP3 mark the transverse and lateral elements of the SC, respectively; CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysfunction, Sclerodactyly, Telangiectasia) marks the centromere and mut L homologue 1 (MLH1, a mismatch repair protein) marks recombination foci, allowing the precise identification of recombination foci along SCs during meiotic prophase. This assay, combined with centromere-specific multicolor fluorescence in situ hybridization (cenM-FISH), allows analysis of recombination distributions of individual chromosomes in human germ cells in great detail (Nietzel et al., 2001; Oliver-Bonet et al., 2003; Sun et al., 2004b, 2006a,b). Using these techniques, we have determined that men with non-obstructive azoospermia have a variety of meiotic defects and a dramatic decrease in the frequency of meiotic recombination (Gonsalves et al., 2004; Sun et al., 2004a, 2005, 2007). These findings have been corroborated in some laboratories (Ma et al., 2006), but not others (Topping et al., 2006). Thus, several lines of evidence point to a relationship between meiotic recombination and aneuploid gametes, but to date, very little research has examined the recombination frequency in spermatogenesis and aneuploid gametes in the same individuals.
In this study, the frequency of meiotic recombination in specific chromosomes was examined in relation to the frequency of sperm aneuploidy for the same chromosomes in the same individuals.
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Materials and Methods |
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Sample collection
Testicular samples were obtained from 11 patients undergoing vasectomy reversal (University of California San Francisco, CA, USA). All of the donors had previously fathered pregnancies. None of the men had any medical illness nor exposure to radiation, chemotherapy or drugs, except one donor, who used ibuprofen as required. Vasectomies had been performed 5–9 years previously. Histological examination showed normal spermatogenesis in all patients (ages 35–61 years). Testicular tissues were kept in phosphate-buffered saline (PBS; pH 7.4) until use, and were shipped on ice to Calgary by air courier. We have previously demonstrated that the cold storage of testicular tissue for 2 days has no effect on the quality of preparations or on data on chromosome pairing or recombination (Sun et al., 2004c
). Ejaculated semen specimens were available from six of the patients (2–26 months later); the rest declined to participate further in the study. Samples were air-freighted on ice to Calgary. Informed consent was obtained from all patients, and this study received ethical approval from the institutional review boards at the University of Calgary and the University of California San Francisco.
Fluorescence immunostaining
Slides with pachytene chromosome spreads were subjected to immunofluorescence staining as described previously (Sun et al., 2004b). Primary antibodies against the following proteins were used: SCP1 (1:1000 dilution, a gift from P. Moens, York University), SCP3 (1:250 dilution, a gift from T. Ashley, Yale University), MLH1 (1:100 dilution, Oncogene, San Diego, CA, USA) and CREST (1:100 dilution, a gift from M. Fritzler, University of Calgary). These primary antibodies were detected using a cocktail of secondary antibodies (donkey antisera) conjugated with different fluorochromes: 1-amino-4-methylcoumarin-3 acetic acid (AMCA) and Cy3 (1:100 dilution, Jackson Immunoresearch, West Grove, PA, USA), AlexaFluor 488 and AlexaFluor 555 (1:125 dilution, Molecular Probes, Eugene, OR, USA). Primary and secondary antibodies were incubated overnight at 37°C, and for 90 min at 37°C, respectively. Slides were examined on a Zeiss Axiophot epifluorescence microscope equipped with rhodamine, fluorescein isothiocyanate (FITC), and 4',6-diamidino-2-phenylindole (DAPI) filters and a cooled charged-coupled device (CCD) camera. Three fluorescent images (red, green and blue) of the SCs, MLH1 sites and CREST locations, respectively, were captured using Applied Imaging Cytovision 3.1 software (Applied Imaging Corporation, Santa Clara, CA, USA). Spreads were localized using a gridded finder slide.
Each pachytene-stage nucleus used for analysis met the following criteria: (i) the correct numbers of bivalents (i.e. 22 autosomes and 1 sex body) were present, (ii) the SCs were not overlapped with other SCs or bent back on themselves, allowing all foci to be scored and (iii) background was fairly low, allowing the SCs to be distinguished from background noise and from each other. MLH1 signals were scored if they were distinct and localized on an SC. SCs were classified as normally synapsed if they were completely linear, without any obvious bubbles, forks, loops or irregularities. One hundred pachytene-stage cells were analyzed for each man, and numbers of MLH1 foci per bivalent and the total number of foci per autosomal complement were scored.
CenM-FISH on spermatocytes
After analysis of the captured immunofluorescence images, cenM-FISH, which allows simultaneous identification of each SC, was carried out on the same spermatocytes, and was used to identify the chromosomes. Techniques developed by Nietzel et al. (2001) and Oliver-Bonet et al. (2003) were modified to make use of the microwave-decondensation/codenaturation FISH technique (Ko et al., 2001). Cells were decondensed for 5 s in dithiothreitol (DTT) and 30 s in 3,5-diiodosalicylic acid, lithium salt (LIS)/DTT at medium power (550 watts). Hybridization buffer (10% dextran sulfate, 2 x standard sodium citrate (SSC), 55% formamide) was prewarmed to 50°C, added to the cenM-FISH probes and warmed at 50°C until all probe was dissolved. Probes were applied to the slide, a glass cover slip was sealed in place with rubber cement, the probes and cells were microwave codenatured for 80 s at 1100 watts and the slide was incubated in a humid chamber at 37°C for 
24 h. A post-hybridization wash (0.4 x SSC, 1% Nonidet P-40; 70°C) was carried out, streptavidin-AlexaFluor 647 (Molecular Probes) solution was applied under a plastic cover slip, and the slide was incubated at 37°C for 40 min in a humid chamber. The slide was washed, with constant agitation, for 10 min in 4 x SSC, air-dried and mounted in DAPI. Cells previously analyzed by antibody immunostaining were relocated, and six fluorescent images (blue, aqua, green, gold, red and far red) were captured for each cell, using Applied Imaging Cytovision 3.1 software (Applied Imaging Corporation, Santa Clara, CA, USA).
After cenM-FISH identification of each pachytene bivalent, the images of corresponding SC spreads were analyzed for MLH1 focus distribution. The numbers of MLH1 foci per bivalent and per SC spread were scored in all males.
FISH on ejaculated sperm
Ejaculated sperm specimens were washed, microwave decondensed and hybridized as described previously (Ko et al., 2001): sperm DNA on the slides was microwave-decondensed with 10 mM DTT (Sigma, Oakville, ON, Canada) (550 watts, 15 s), followed by 10 mM LIS (Sigma)/1 mM DTT (550 watts, 1.5 min). Slides were rinsed, air-dried at room temperature, dehydrated in 80% methanol at –20°C for 20 min, air-dried, and used immediately. Sex chromosome hybridizations were carried out using a Fluorogreen-TM (Amersham, Baie d'Urfé, QC, Canada) labeled X-specific 
-satellite probe, kindly provided by E. Jabs of the Johns Hopkins University, Baltimore, MD, USA (Jabs et al., 1989), a Fluoroblue-TM labeled chromosome 1-specific satellite III sequence, pUC1.77, generously provided by H.J. Cooke of Edinburgh, Scotland (Cooke and Hindley, 1979) and a CEP SpectrumOrange Yq probe (Vysis, Downer's Grove, IL, USA). Chromosome 1/9 hybridizations were carried out using the same chromosome 1-specific satellite III sequence labeled directly with FluorogreenTM, and a CEP SpectrumOrange 9 probe (Vysis). Chromosome 13/21 hybridizations used SpectrumGreen 13 LSI and SpectrumOrange 21 LSI probes (Vysis).
Scoring of sperm nuclei
Slides were counted using a Zeiss Axiophot microscope fitted with four filter sets: FITC, rhodamine/FITC, DAPI and rhodamine/FITC/DAPI. Two same-colored signals were counted as individual signals if they were separated by at least one signal diameter (1/2 signal diameter for the overlarge Y signal) and were of similar size, shape and intensity. The blue chromosome 1 signal in sex chromosome hybridizations was used as an internal autosomal control to distinguish between disomy and diploidy.
Statistical analysis
Correlations between the recombination frequency and frequencies of aneuploidy in chromosomes were performed using Pearson and Spearman correlation analyses. One-way analysis of variance was used to determine if chromosome 21 and the sex chromosomes had a higher frequency of bivalents with no recombination compared with other chromosomes. A value of P < 0.05 was considered significant.
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Results |
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An example of pachytene SCs, with identification of individual bivalents and cenM-FISH signals in the same cell, is shown in Fig. 1. A total of 591 pachytene stage spreads with unambiguous cenM-FISH signals were analyzed to determine the MLH1 focus frequency for chromosomes 1, 9, 13 and 21 in the six men who provided both testicular samples and ejaculated sperm. On average, the mean (±SD) frequency of MLH1 foci per cell for chromosomes 1, 9, 13 and 21, was 3.42 ± 0.80, 2.01 ± 0.60, 1.67 ± 0.51 and 0.86 ± 0.27, respectively (Table I). In all, 600 pachytene-stage spermatocytes (100 cells for each male) were analyzed to determine the mean MLH1 focus frequency per cell for autosomes, with an overall mean (±SD) of 50.7 ± 4.7 foci (range: 32–63) (Table I). On average, there was an MLH1 focus in the sex body of 86/100 cells (range: 80–91) (Table I). These frequencies of recombination foci in autosomes and sex chromosomes are very similar to those in previous reports (Gonsalves et al., 2004
; Codina-Pascual et al., 2006; Sun et al., 2006b). The frequency of bivalents with 0 MLH1 foci is presented in Table II.
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The sperm aneuploidy frequency for chromosomes 1, 9, 13, 21, X and Y was assessed by FISH analysis. About 10 000 spermatozoa were scored for each donor (Table III). No difference between the disomy frequencies for XX and YY was observed (both were 0.03%). However, the XY disomy frequency (0.17%) was higher than the combined values of XX and YY disomy (0.06%). The highest autosomal disomy and nullisomy frequencies were both observed in chromosome 21.
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Correlation analysis between the frequency of recombination foci (calculated as MLH1 focus frequency per specific chromosome) and of sperm chromosome aneuploidy in the same individuals was performed to examine the possibility of a direct association between meiotic recombination and sperm aneuploidy in humans. No significant correlation was found between the mean frequency of cells with a sex body containing an MLH1 focus and XX, YY or XY disomy in ejaculated sperm, nor between the mean MLH1 frequency for chromosomes 1, 9, 13 and 21 and aneuploidy for the corresponding chromosome (P > 0.05, Pearson correlation analysis). An example of this relationship in chromosome 21 (correlation coefficient r = –0.1185, P = 0.8230, Pearson correlation analysis) is shown in Fig. 2. Also, there was no significant correlation between the frequency of bivalents with no recombination foci and the frequency of sperm aneuploidy (P > 0.05, Spearman correlation analysis).
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Chromosome 21 had a higher frequency of bivalents with no recombination foci compared with the other autosomes (P < 0.0001) and the sex chromosomes had a higher frequency than any other chromosome (P < 0.0001).
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Discussion |
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We originally observed, by both human sperm karyotype studies (Martin and Rademaker, 1990
) and FISH analysis (Spriggs et al., 1996; Shi and Martin, 2000), that the G group chromosomes (21 and 22) and the sex chromosomes had a significantly higher frequency of sperm aneuploidy than other chromosomes. This research was corroborated by other laboratories (Williams et al., 1993; Blanco et al., 1998). This led us to hypothesize that the reason might be that these chromosomes normally only have one crossover: chromosomes 21 and 22 being the smallest chromosomes and the sex chromosomes pairing and recombining only in the pseudoautosomal region. If this single crossover were lost, a dangerous situation would ensue, since there would be no mechanism to ensure proper segregation of homologous chromosomes into daughter cells. Our first study on recombination in sex chromosomes by single sperm typing demonstrated a decrease in recombination in the pseudoautosomal region in sperm that had undergone non-disjunction compared with normal sperm (Shi et al., 2001). Furthermore, immunofluorescence analysis on pachytene cells in 10 normal men demonstrated that chromosomes 21 and 22 had a higher frequency of non-crossover bivalents compared with other autosomes, and that the sex chromosomes had the highest frequency of univalents overall (Sun et al., 2006a), results that have since been corroborated (Codina-Pascual et al., 2006). Thus our expectations were fulfilled—that the chromosomes with the highest frequency of achiasmate bivalents were the same chromosomes that had the highest frequency of sperm aneuploidy. However, a direct association between meiotic recombination and sperm aneuploidy in the same individual, which would more accurately reflect how faulty recombination effects might directly impact sperm aneuploidy, was lacking. This study provides that missing link.
In this study, the frequency of meiotic recombination in chromosomes 1, 9, 13, 21, X and Y was compared with the frequency of sperm aneuploidy for the same chromosomes in six men. Of the chromosomes studied, it is clear that the sex bivalent and chromosome 21 have the highest frequency of bivalents with no recombination foci (Table II). However, these same chromosomes do not have a significantly higher frequency of aneuploidy in sperm compared with the other chromosomes (Table III). This is puzzling, since we have observed a higher frequency of sperm aneuploidy for the G group and sex chromosomes in many studies in the past, including in healthy (Martin and Rademaker, 1990; Spriggs et al., 1996) and infertile men (Martin et al., 2003b). However, these fertile vasectomy reversal patients are a more select uniform population of proven fertility, and comparison of their frequencies of sperm aneuploidy with previous healthy populations demonstrates a lower mean frequency of disomy for both the sex chromosomes [0.23 versus 0.41 in previous studies (Kinakin et al., 1997)] and chromosome 21 [0.24 versus 0.37 (McInnes et al., 1998)]. It is comforting to know that men seeking vasectomy reversal do not appear to have sperm with elevated frequencies of aneuploidy despite years of physical obstruction of the excurrent ducts in the reproductive tract.
Codina-Pascual et al. (2005) studied infertile men and found a relationship between a low frequency of recombination in the sex body and the overall frequency of recombination in the autosomes. We found no such relationship, but as can been seen from Table I, these fertile men had a uniformly high percentage of cells with a recombination focus in the sex chromosomes, demonstrating the normality of the meiotic process in these men.
To date, there have been no other studies comparing meiotic recombination frequency with sperm aneuploidy frequencies in ejaculated spermatozoa. However, Ma et al. (2006) studied one infertile man and found that a total lack of recombination in the meiotic sex chromosomes was mirrored by a high total sex chromosome aneuploidy frequency in testicular sperm. Also, a reduced recombination frequency for chromosomes 13 and 21 was associated with an increased frequency of aneuploidy in testicular sperm. It is possible that they found a significant association between lack of recombination and sperm aneuploidy because their one infertile patient had an exceptionally low frequency of recombination (0% in the sex chromosomes) coupled with a very high frequency of sex chromosome aneuploidy (41.6%). Our values in proven fertile men were much less extreme, with most chromosome bivalents having at least one recombination focus. Thus, our fertile men may not have reached the threshold required to demonstrate the relationship between recombination and aneuploidy. Certainly, our studies and those of others have demonstrated that infertile men with non-obstructive azoospermia have a decreased frequency of meiotic recombination (Gonsalves et al., 2004; Sun et al., 2004a, 2005, 2007) and an increased risk of aneuploidy (Palermo et al., 2002; Martin et al., 2003a). However, although these studies compared testicular tissue and ejaculated sperm, the comparison was not made in the same individuals. The approach applied here provides a unique avenue to investigate the association of human meiotic events with aneuploidy more precisely, and has the potential to provide important information on the basic mechanisms underlying non-disjunction in humans and also to shed light on the cause of the increased frequency of chromosome abnormalities in infertile men. It will be important to study infertile men with oligozoospermia or azoospermia in a similar manner, to determine if our hypothesis of a threshold effect on a correlation between meiotic recombination and sperm aneuploidy exists.
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Funding |
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R.H.M. holds the Canada Research Chair in Genetics, and the research was funded by the Canadian Institutes of Health Research (CIHR) grant MA7961. F.S. and M.O-B. are the recipients of a CIHR Strategic Training Fellowship in Genetics, Child Development and Health.
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Submitted on October 15, 2007; resubmitted on November 26, 2007; accepted on January 18, 2008.
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