Skip Navigation


Human Molecular Genetics Advance Access originally published online on August 29, 2007
Human Molecular Genetics 2007 16(23):2870-2879; doi:10.1093/hmg/ddm246
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/23/2870    most recent
ddm246v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ferguson, K. A.
Right arrow Articles by Ma, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ferguson, K. A.
Right arrow Articles by Ma, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy

Kyle A. Ferguson, Edgar Chan Wong, Victor Chow, Mark Nigro and Sai Ma*

Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, Canada V6H 3N1

* To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Room D414B, BC Women's Hospital and Health Centre, D6-4500 Oak Street, Vancouver, British Columbia, Canada V6H 3N1. Tel: +1 6048752345 ext: 5686; Fax: +1 6048752722; Email: sai{at}interchange.ubc.ca

Received July 25, 2007; Accepted August 20, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Defects in early meiotic events are thought to play a critical role in male infertility; however, little is known regarding the relationship between early meiotic events and the chromosomal constitution of human sperm. Thus, we analyzed testicular tissue from 26 men (9 fertile and 17 infertile men), using immunofluorescent techniques to examine meiotic chromosomes, and fluorescent in situ hybridization to assess sperm aneuploidy. Based on a relatively small sample size, we observed that 42% (5/12) of men with impaired spermatogenesis displayed reduced genome-wide recombination when compared to the fertile men. Analysis of individual chromosomes showed chromosome-specific defects in recombination: chromosome 13 and 18 bivalents with only a single crossover and chromosome 21 bivalents lacking a crossover were more frequent among the infertile men. We identified two infertile men who displayed a novel meiotic defect in which the sex chromosomes failed to recombine: one man had an absence of sperm in the testes, while the other displayed increased sex chromosome aneuploidy in the sperm, resulting in a 45,X abortus after intracytoplasmic sperm injection. When all men were pooled, we observed an inverse correlation between the frequency of sex chromosome recombination and XY disomy in the sperm. Recombination between the sex chromosomes may be a useful indicator for identifying men at risk of producing chromosomally abnormal sperm. An understanding of the molecular mechanisms that contribute to sperm aneuploidy in infertile men could aid in risk assessment for couples undergoing assisted reproduction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Improper segregation of chromosomes during meiosis can result in the production of genetically unbalanced sperm or oocytes. If these gametes participate in fertilization, the resulting embryo will be aneuploid, with either too many chromosomes (trisomy) or too few (monosomy). Aneuploidy is the most common chromosomal abnormality in humans, occurring in 5% of all pregnancies and 0.3% of livebirths (1). Although the majority of numerical chromosomal abnormalities are of maternal origin, paternal errors account for the majority of sex chromosome aneuploidies. Prenatal testing of intracytoplasmic sperm injection (ICSI) pregnancies has shown an increase in de novo sex chromosome abnormalities (2), the majority of which are of paternal origin (3). Studies on the chromosomal constitution of sperm from infertile men have shown that this population may be at an increased risk of producing aneuploid sperm (4–6). Thus, the increased aneuploidy in the sperm of infertile men is likely one of the major sources of chromosomal abnormalities in ICSI pregnancies, and the transmission of these aneuploidies may be facilitated via ICSI (7).

During the first meiotic division, homologous chromosomes must undergo synapsis, in which the chromosomes pair-up and a protein structure known as the synaptonemal complex (SC) forms between the two homologs. It is along this protein structure that meiotic recombination occurs. Studies have identified aberrant meiotic recombination as an important molecular factor causing meiotic non-disjunction (8–11). Meiotic recombination not only serves to generate genetic diversity, but crossovers also tether homologous chromosomes, thus facilitating the proper segregation of chromosomes during meiosis. Abnormalities in the frequency and location of crossovers are associated with non-disjunction of homologous chromosomes and the production of aneuploid gametes (10,12,13). Thus, the increased frequency of chromosomal abnormalities in the sperm of infertile men may be the result of defects in critical meiotic events during spermatogenesis. Defects in synapsis or recombination may be caught by meiotic checkpoints, leading to a loss of germ cells and subsequent infertility (14–16). However, some cells may be able to progress through meiosis, resulting in an increased proportion of sperm with chromosomal abnormalities.

Using antibodies against SC protein 3 (SCP3) (axial elements) and SCP1 (transverse elements) to visualize the SC, antibodies against Mut-L homologue 1 (MLH1) for localization of crossovers, and CREST antiserum to observe the centromeres of chromosomes, detailed meiotic events can now be studied in human males (17,18). Recent immunofluorescent studies have shown that defects in synapsis and recombination are a significant cause of male infertility (19–22). Nevertheless, these studies have not addressed whether infertile men showing defective synapsis and recombination are at an increased risk of producing aneuploid sperm. Furthermore, there is little information on the chromosome-specific patterns of recombination in infertile men. Thus, we combined immunofluorescent techniques and fluorescent in situ hybridization (FISH) on spermatocytes to study recombination and synapsis in infertile men. Chromosomes 13, 18, 21 and the sex chromosomes were studied specifically, as aneuploidies of these chromosomes are a major cause of spontaneous abortions and abnormalities in liveborns. FISH on testicular sperm from these same men was performed to determine if certain meiotic phenotypes were particularly at risk of producing aneuploid sperm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Classification of subgroups
Testicular tissue was collected and analyzed from 26 men seeking fertility treatment. These men were classified into four groups. Group I was the control group, consisting of nine fertile men who had undergone a vasectomy after fathering a child naturally. The remaining men presented at the fertility clinic with idiopathic forms of infertility, and were classified into groups based on their pathological diagnosis. Four standard pathological diagnoses for spermatogenesis were used: normal spermatogenesis, hypospermatogenesis (a reduced number of germ cells showing normal maturation), maturation arrest (germ cells present up to a certain stage) and Sertoli cell only (a lack of germ cells). The 17 infertile men were divided into three groups: group II [obstructive azoospermia (OA)], men diagnosed with normal spermatogenesis with no sperm in the semen (n = 5); group III [non-obstructive azoospermia (NOA)], men with abnormal spermatogenesis without sperm in the semen (n = 10), including six men diagnosed with either hypospermatogenesis or maturation arrest, and four men with Sertoli cell only syndrome; and group IV [oligoasthenoteratozoospermia (OAT)], men with a very low sperm count in the ejaculate, combined with abnormal sperm morphology and motility (n = 2).

Progression through prophase I in infertile men
Prophase cells were classified as leptotene, zygotene, zygotene/pachytene or pachytene, according to criteria previously described (23). The mean frequencies of cells in the control group at the leptotene, zygotene, zygotene/pachytene and pachytene stages were 9.9, 1.5, 8.1 and 80.4%, respectively (Table 1). There was no significant difference in the proportion of cells at any of the stages of prophase between the control group and the OA group. However, the NOA and OAT groups showed a significantly increased proportion of cells at the leptotene stage, as well as a significantly decreased proportion of cells at the pachytene stage, when compared to the control group (P < 0.001, Mann-Whitney test; Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Analysis of the progression through prophase I in fertile and infertile men

 
Analysis of genome-wide recombination
Immunostaining of spermatocytes with antibodies to mark sites of recombination (MLH1) and the SC (SCP1 and SCP3) allowed us to assess rates of recombination and synaptic errors in spermatocytes of fertile and infertile men (Fig. 1A). A total of 605 pachytene nuclei were analyzed from the control group, and 1057 pachytene nuclei from the three infertile groups. The control group showed an average of 47.6 ± 2.2 crossovers per cell, with individual mean rates of recombination ranging from 44.3 ± 5.7 to 51.3 ± 5.8 (Table 2). The recombination levels that we observed in our control men are within those reported by others (18,24,25). The average recombination rate was 46.4 ± 2.5 (range: 42.1 ± 4.7 to 48.2 ± 3.5) in the OA group, 43.7 ± 5.9 (range: 34.0 ± 3.0 to 50.2 ± 5.3) in the NOA group and 33.9 ± 6.2 in the OAT group. When the means of the individuals were pooled and compared to the control group, only the OAT group was significantly different (P < 0.05, Mann-Whitney test). However, there was significant inter-individual variation among men in the OA and NOA groups. One man in the OA group (OA14) showed significantly reduced recombination when compared to C9, the control man displaying the lowest average recombination rate (P < 0.01, Mann-Whitney test). Five men (NOA10, NOA13, NOA 16, OAT1, OAT8) in the NOA and OAT groups displayed reduced recombination when compared to C9 (P < 0.01, Mann-Whitney test). However, recombination was only significantly reduced in three of the men (NOA13, OAT1, OAT9) from the NOA and OAT groups when compared to the individual with the lowest mean in the OA group (OA14) (P < 0.01, Mann-Whitney test).


Figure 1
View larger version (92K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Immunofluorescent and FISH analysis of pachytene nuclei. (A) Spermatocytes were immunolabeled to visualize the SC (red), MLH1 (green) and centromeres (blue). (B) Subsequent FISH on the same spermatocytes allowed us to characterize recombination on chromosome 13 (green), chromosome 18 (blue) and chromosome 21 (red). In (A), the chromosome 21 bivalent (arrow) and XY bivalent (arrowhead) display no recombination. Other meiotic defects were observed, including (C) unsynapsed regions of the SC (arrows); and (D) fragmentation of the SC, which was observed in one infertile man (OAT8).

 


View this table:
[in this window]
[in a new window]

  		Table 2. Analysis of MLH1 foci and synaptic errors in fertile and infertile men

 
The proportion of pachytene cells with a crossover in the sex body ranged from 71.0 to 92.7% in the control group, and 73.0 to 93.2% in the OA group (Table 2). However, recombination between the sex chromosomes was particularly disrupted in the NOA and OAT groups, with four of the men (NOA10, NOA13, OAT1, OAT8) displaying significantly reduced recombination when compared to the lowest control (P < 0.001, Fisher test). Two of these men (NOA10, OAT1) showed no recombination between the sex chromosomes in any of the pachytene nuclei observed. This absence of recombination between the X and Y chromosomes has not been observed in other meiotic studies on infertile men (19–22). Previous meiotic studies on azoospermic men have also observed impaired recombination on autosomal chromosomes, with an increase in pachytene nuclei in which at least one autosomal chromosome is lacking a crossover (19,22). We also observed a similar increase in pachytene cells containing at least one achiasmate autosomal chromosome in the NOA and OAT groups (Table 2). Five of these men (NOA10, NOA13, NOA15, OAT1, OAT8) displayed a significant increase in pachytene nuclei with at least one autosomal chromosome lacking a crossover, when compared to all of the men in the control group (range: 0–5.9%) and OA group (range: 1.0–6.0%) (P < 0.001, Fisher test).

Synaptic anomalies
Two types of synaptic anomalies were observed during pachytene analyses in all groups of men: (i) discontinuities in the SC, in which both the lateral (SCP3) and transverse (SCP1) elements were absent in regions of the SC, and (ii) unsynapsed regions of the SC in which meiotic pairing of the homologous chromosomes was incomplete, and only the lateral elements (SCP3) were present (Fig. 1C). Discontinuities in the SC were the more common of the synaptic anomalies, with a mean frequency of 26.6% (range: 5.9–56.1%) in the control group, although high interindividual variation was observed (Table 2). The frequency of SC discontinuities was also highly variable in the OA group (range: 16.4–57.0%), as well as the NOA group (range: 11.5–22.0%). Unsynapsed meiotic chromosomes were less frequent than discontinuities, with a mean frequency of 3.3% (range: 0–9.0%) in the control group (Table 2). One man in the OA group (OA9) showed a significant increase in unsynapsed chromosomes when compared to all of the control men, with 26.0% of pachytene nuclei displaying this synaptic anomaly (P < 0.05, Fisher test). Unlike other studies (19,22), we did not identify any men in the NOA or OAT groups who displayed rates of unsynapsed regions that were significantly greater than all of the control men. However, we did identify one man (OAT8) in which synaptic anomalies could not be accurately assessed, as fragmentation of the SC was observed in almost all pachytene nuclei (Fig. 1D).

Chromosome-specific recombination frequencies and sperm aneuploidy
After observing that genome-wide recombination rates were reduced in some of the infertile men, we were interested in analyzing chromosome-specific patterns of recombination. The combination of FISH and immunofluoresent techniques enabled us to characterize the frequency of recombination on chromosomes 13, 18 and 21 (Figs. 1A and B and Table 3). From the control group, we analyzed 338 pachytene nuclei from 6 men, and from the infertile groups we analyzed 591 pachytene nuclei from 9 men. We observed no significant differences in the chromosome-specific frequencies of recombination between the control group and the OA group. However, chromosome-specific recombination frequencies appeared to be altered in the NOA and OAT groups. In the control group, chromosome 13 displayed two MLH1 foci in 83.4% of the pachytene nuclei, which was significantly greater than both the 40.6% in the pooled NOA group and 46.0% in the OAT1 patient (P < 0.001, Fisher test). Similarly, chromosome 18 displayed two MLH1 foci in 69.8% of pachytene nuclei from the control group, which was significantly greater than the 32.7% of pachytene nuclei from the pooled NOA men and 38.1% from OAT1 (P < 0.001, Fisher test). Chromosome 21 was achiasmate in 4.0% of nuclei from the pooled NOA group and 10.6% from OAT1, which were both significantly greater than the 0.61% of pachytene nuclei from the control men (P < 0.001, Fisher test). Two infertile men (NOA13, OAT1) showed a significant increase in the frequency of achiasmate chromosome 13 when compared to the control group (P < 0.005, Fisher test); however, no increase in the frequency of achiasmate chromosome 18 was observed in any of the infertile men.


View this table:
[in this window]
[in a new window]

  		Table 3. Analysis of crossovers on chromosomes 13, 18 and 21 in fertile and infertile men

 
Recombination is thought to play a critical role in ensuring the proper disjunction of chromosomes during meiosis. Thus, we were interested in determining if the infertile men with altered recombination rates were at an increased risk of producing chromosomally abnormal sperm. To assess sperm aneuploidy, we performed triple-color FISH for chromosomes 18, X and Y, followed by dual-colour FISH for chromosomes 13 and 21 on sperm extracted from the testicular tissue samples. A total of 22 547 sperm were scored from the control men, and 18 634 sperm from the three infertile groups. No sperm could be found in the testes in two of the infertile men with meiotic cells (NOA10, NOA13), and thus aneuploidy could not be assessed in these men. The mean frequency of disomy in sperm from the control, OA, NOA groups, respectively, were 0.21, 0.31 and 0.23% for XX and YY; 0.25, 0.51 and 0.84% for XY; 0.21, 0.38 and 0.19% for chromosome 13; 0.10, 0.26 and 0.23% for chromosome 18; and 0.40, 0.36 and 0.38% for chromosome 21 (Table 4). Thus, aneuploidy rates were only modestly increased for XY disomy in the NOA group when compared to the control group. Surprisingly, the OA group also showed a modest increase in XY disomy in the sperm, although the rate was significantly lower than the NOA group (P < 0.05, Fisher test). However, the two men with OAT displayed much higher disomy rates for the sex chromosomes and all autosomes studied (Table 4). OAT1 showed the most dramatically increased sperm aneuploidy of all the men studied, particularly for XY disomy which was present in 25.24% of the sperm.


View this table:
[in this window]
[in a new window]

  		Table 4. Testicular sperm aneuploidy rates in fertile and infertile men

 
When all of the men (control and infertile) were combined, we observed an inverse correlation between the frequency of recombination between the sex chromosomes and XY disomy in the sperm, with sperm disomy rates dropping as recombination increased (P < 0.001, r = –0.75, Pearson's test). We thought that two of the infertile men (OAT1, OAT8) with the most disrupted XY recombination may have been skewing the results; however, a relationship was still observed even when these two men were excluded (P = 0.033, Pearson's test, Fig. 2). Next, we wanted to see if there was a relationship between chromosome-specific recombination on autosomal chromosomes and sperm disomy rates. When we combined all of the men in which chromosome-specific rates of recombination were studied (Table 3), we observed a significant relationship between the frequency of achiasmate chromosome 21 and disomy 21 in the sperm (P < 0.001, r = 0.89, Pearson's test). However, the OAT1 patient appeared to be an outlier in this group, and when he was eliminated no significant relationship was observed (P = 0.29, r = 0.32, Pearson's test). Similarly, no relationships were observed between rates of recombination and disomy rates in the sperm for chromosomes 13 and 18.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Relationship between the frequency of XY recombination and XY disomy in sperm from 18 fertile and infertile men. Rates of XY disomy were highly variable among men, and appear to be inversely correlated with the frequency of XY recombination in spermatocytes (P = 0.033, r = –0.49). Two men (OAT1, OAT8) were not included in the correlation due to their extremely low levels of XY recombination.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
A series of recent immunofluorescent studies on infertile men have shown that meiotic defects are routinely observed among infertile men (19–22). In this study, we set out to further characterize the meiotic defects that have been observed in the infertile population, as well as to investigate chromosome-specific deficiencies in recombination. Furthermore, we were interested in determining if abnormalities in recombination were associated with the production of chromosomally abnormal sperm.

Two previous studies have identified infertile men showing complete arrest at the zygotene stage, in which no pachytene cells could be observed, due to severe synaptic defects (19,21). We did not observe this phenotype in any of our infertile men; however, when we classified meiotic cells into different stages of prophase, we observed an increase in the proportion of cells at the leptotene stage and a decrease in the proportion of cells at the pachytene stage among men with NOA or severe OAT. This impaired progression through meiosis has also been observed in another study on infertile men (22). Defective formation or synapsis of the SC leads to male infertility in mice (16), possibly through the apoptotic cell death of pachytene cells by stringent meiotic checkpoints (26).

Defects in recombination and synapsis in infertile men
The development of immunofluorescent techniques for the analysis of pachytene cells has provided an efficient method for assessing meiotic recombination frequencies in males (17). The initial studies performed on normal males have provided a baseline of recombination frequencies in males (18,24,25), and more recent studies have identified several infertile men displaying reduced genome-wide levels of recombination (19,22). These studies have confirmed early cytological observations that defects in both meiotic recombination (27,28) and synapsis (29) are associated with male infertility. In this study, we have identified six infertile men (OA14, NOA10, NOA13, NOA15, OAT1, OAT8) who displayed reduced levels of genome-wide recombination when compared to all of the control men, thus confirming the observations of Gonsalves et al. (19) that recombination deficiencies are a significant factor in male infertility. Interestingly, we identified two men (NOA10, OAT1) who showed a novel recombination deficiency: a complete absence of recombination between the sex chromosomes in all pachytene cells analyzed. While other studies have identified infertile men displaying reduced recombination between the sex chromosomes (19,20,22), all of the men previously examined showed some degree of XY recombination. The lack of recombination between the sex chromosomes had differing effects on the outcome of spermatogenesis, with one man (NOA13) showing an absence of sperm in the testes, while the other man (OAT1) produced a low number of sperm with extremely elevated rates of sperm aneuploidy. Thus, XY-recombination may be necessary for the completion of spermatogenesis in some men (30), however other men may display less stringent checkpoints and be capable of completing spermatogenesis. While several gene knock-out mice have been found to display altered recombination patterns (31–33), none display the lack of XY-recombination that we observed.

Synaptic errors, such as unpaired regions or gaps in the SC, did not appear to be a major contributing factor to infertility in our infertile population. None of the men with NOA or OAT displayed increased rates of synaptic errors when compared to all of the control men. These observations were in contrast to those of Sun et al. (22). who observed an increase in synaptic errors in their NOA population. Surprisingly, the only man displaying a significant increase in unpaired regions of the SC was OA9, whose histological diagnosis reported normal spermatogenesis. Unpaired regions were found in 26.0% of pachytene nuclei from this man, and thus a sufficient proportion of spermatocytes may have been able to progress through meiosis for spermatogenesis to appear unaffected. Unpaired regions of meiotic chromosomes are transcriptionally silenced in the mouse through the recruitment of BRCA1 and {gamma}H2AX (34,35), and may also trigger meiotic checkpoints (36), and have therefore been suspected to play a role in some cases of infertility. Fragmentation of the SC and low levels of meiotic recombination were observed in most pachytene nuclei from one man (OAT8), which may be indicative of spermatocytes undergoing apoptotic cell death. The SC fragmentation that we observed in this man was similar to that observed in fetal oocytes thought to be at risk of atresia (37) and spermatocytes from men with AZFc deletions on the Y chromosome (38).

Recombination and sperm aneuploidy
Recently, several studies have combined immunofluorescent and FISH analysis of pachytene nuclei to characterize the recombination patterns on specific chromosomes of normal males (39–41). Nevertheless, there is no information on the chromosome-specific frequencies of recombination in the infertile population. Recombination in the NOA and OAT groups appeared to be reduced on all three of the autosomal chromosomes studied. Chromosome 13 and 18 bivalents with only one crossover were more prevalent in the NOA and OAT groups when compared to the control group, who displayed double crossovers on the majority of bivalents. Furthermore, a greater proportion of chromosome 21 bivalents lacked a crossover in the NOA and OAT groups. The achiasmate chromosome 21 bivalents may be caught by a checkpoint and induce meiotic arrest (26); however, they may also increase the risk of meiotic non-disjunction and trisomy 21 in the offspring (10).

In both model organisms and humans, recombination is thought to be essential for the proper segregation of chromosomes during meiosis (42). Thus, the reduced levels of both genome-wide and chromosome-specific recombination that have been observed in some infertile men may increase their risk of producing aneuploid sperm and chromosomally abnormal offspring. Furthermore, the variation in recombination parameters among both fertile and infertile men may explain the inter-individual variation in sperm aneuploidy rates (43). By performing FISH on testicular sperm, we were able to assess disomy rates for chromosomes 13, 18, 21 and the sex chromosomes. The modest increase in XY disomy in testicular sperm from men with NOA compared to normal controls is consistent with other reports (44–46); however, we were surprised to find that the OA population also displayed an increased rate of XY disomy in the sperm. It appears that men diagnosed with OA may nevertheless display meiotic abnormalities, such as an increased frequency of unsynapsed meiotic chromosomes (OA11) or a decreased frequency of recombination (OA14), which may increase the risk of aneuploid sperm. It should be noted that, unlike other studies (19), our group of OA men had no obvious blockages in the seminal tract, such as a congenital absence of the vas deferens. Thus, although a histological analysis of spermatogenesis may appear normal, subtle meiotic defects may be present in both the spermatocytes and sperm of men who are considered OA.

While we found no correlation between rates of recombination and sperm disomy for chromosomes 13, 18 and 21, we found an inverse correlation between rates of sex chromosome recombination and XY disomy in the sperm. Thus, men with higher rates of recombination between the sex chromosomes face a smaller risk of producing XY disomic sperm. This supports previous observations that most paternally derived 47,XYY males were conceived by sperm in which the sex chromosomes did not undergo meiotic recombination (9). This suggests that men with extremely defective XY recombination may be at a greater risk of fathering offspring with a sex chromosome abnormality. Indeed, an attempt at ICSI using sperm from an infertile man who displayed 0% recombination between the sex chromosomes (OAT1) resulted in a 45,X abortus of paternal origin (23).

In this study, we have contributed to the growing evidence that meiotic defects are a significant cause of male-factor infertility. By examining both the early meiotic events in spermatocytes and the chromosomal constitution of the sperm in the same men, we found that recombination between the sex chromosomes was inversely correlated with XY disomy rates in the sperm. XY recombination rates are variable among men, and this may explain both the inter-individual variation in sperm aneuploidy rates, as well as the increased sperm aneuploidy rates that have been frequently reported among the infertile population. We also identified a novel meiotic defect in two unrelated men, in which no recombination was observed between the sex chromosomes. In one case, spermatogenic arrest and an absence of sperm in the testes was observed. However, the other case produced sperm with extremely elevated rates of sex chromosome aneuploidy, which resulted in a 45,X abortus after ICSI. This is the first evidence that defective recombination in infertile men may be associated with elevated levels of sperm aneuploidy, and chromosomally abnormal offspring. Further meiotic studies on infertile men will provide important insights into the genetic basis of idiopathic male infertility, and could also provide useful information for predicting rates of sperm aneuploidy, which could aid in risk assessment for couples undergoing assisted reproduction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Patients and issue collection
Testicular tissue was collected and analyzed from 17 infertile men seeking fertility treatment and 9 proven fertile men who were undergoing vasectomy reversals. All infertile men presented at the fertility clinic with idiopathic forms of infertility, and had normal 46,XY karyotypes, no microdeletions on the Y chromosome, and no CFTR mutations. The testicular tissue was used for a pathology diagnosis, sperm extraction for subsequent ICSI cycles, and a small portion was used for the meiotic analyses reported in this study. Cases NOA4, NOA5, OA6, OAT1, C1 and C3 were reported in previous publications (23,47). Ethical approval was obtained from the University of British Columbia Ethics Committee before initiating this study.

Meiotic analyses
Testicular tissue was processed as previously reported (47). Slides were scanned with a Zeiss Axioplan epifluorescent microscope equipped with appropriate filters. Images of the SCP3/SCP1 fragments, MLH1 and CREST sites were captured using Cytovision V2.81 Image Analysis software (Applied Imaging International, San Jose, CA, USA). Pachytene cells were captured if MLH1 foci were clear and the sex chromosomes were identifiable. Cell co-ordinates were recorded and prints were analyzed for the numbers of MLH1 and abnormalities in the SC.

FISH on spermatocytes and testicular spermatozoa
After capturing images of the SC and MLH1 foci, FISH was performed on the same spermatocytes to identify chromosomes 13, 18 and 21 as previously described (23). Slides of testicular cells containing sperm which were not previously immunostained, were used for FISH analysis of testicular sperm aneuploidy. FISH on spermatozoa was performed as previously reported (7). A probe mixture of CEP 18 (aqua)/CEP X (green)/CEP Y (red) was hybridized to sperm (Vysis Inc., Downers Grove, IL, USA). The same slide was then re-hybridized with a probe mixture of LSI 13 (green)/LSI 21 (red). Spermatozoa were only counted if they had intact tails and were not overlapping with other cells. We attempted to score at least 1000 sperm per patient for each probe set.

Statistical analysis
The Mann–Whitney test was used to compare means between groups, as well as the mean rates of recombination between individuals. The Fisher exact test was used to compare the proportion of cells in difference stages of prophase, the proportion of cells with synaptic anomalies and the frequency of recombination on specific chromosomes. The Pearson's correlation test was used to identify a relationship between recombination rates and sperm disomy rates.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
The Canadian Institute of Health Research (MOP53067 to S.M.). K.A.F is the recipient of the Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada, and the Junior Graduate Studentship from the Michael Smith Foundation for Health Research.

Conflict of Interest statement. None declared.


   ACKNOWLEDGEMENTS
 
We thank the patients for donating samples, as well as the clinical and laboratory staff of the University of British Columbia IVF program in the Division of Reproductive Endocrinology and Infertility for their assistance in this study. We gratefully thank Dr P. Moens for providing the antibodies.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

  1. Hassold T., Abruzzo M., Adkins K., Griffin D., Merrill M., Millie E., Saker D., Shen J., Zaragoza M. Human aneuploidy: incidence, origin and etiology. Environ. Mol. Mutagen. (1996) 28:167–175.[CrossRef][Web of Science][Medline]

  2. Bonduelle M., Van Assche E., Jordis H., Keymolen K., Devroey P., Van Steirteghem A., Liebaers I. Prenatal testing in ICSI pregnancies: incidence of chromosomal abnormalities in 1586 karyotypes and relation to sperm parameters. Hum. Reprod. (2002) 17:2600–2614.[Abstract/Free Full Text]

  3. Van Opstal D., Los F.J., Ramlakhan S., Van Hemel J.O., Van Den Ouweland A.M.W., Brandenburg H., Pieters M.H.E., Verhoeff A., Vermeer M.C.S., Dhont M., et al. Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum. Reprod. (1997) 12:682–686.[Abstract/Free Full Text]

  4. Pang M.G., Hoegerman S.F., Cuticchia A.J., Moon S.Y., Doncel G.F., Acosta A.A., Kearns W.G. Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection. Hum. Reprod. (1999) 14:1266–1273.[Abstract/Free Full Text]

  5. Moosani N., Pattinson H.A., Carter M.D., Cox D.M., Rademaker A.W., Martin R.H. Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril. (1995) 64:811–817.[Web of Science][Medline]

  6. Bernardini L., Martini E., Garaedts J.P., Hopman A.H., Lanteri S., Conte N., Capitanio G.L. Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol. Hum. Reprod. (1997) 3:431–438.[Abstract/Free Full Text]

  7. Tang S.S., Gao H., Robinson W.P., Ho Yuen B., Ma S. An association between sex chromosomal aneuploidy in sperm in an abortus with 45,X of paternal origin: possible transmission of chromosomal abnormalities through ICSI. Hum. Reprod. (2004) 19:147–151.[Abstract/Free Full Text]

  8. Warren A.C., Charkravarti A., Wong C., Slaugenhaupt S.A., Halloran S.L., Watkins P.C., Metazotou C. Evidence for reduced recombination on the nondisjoined chromosome 21 in Downe syndrome. Science (1987) 237:652–654.[Abstract/Free Full Text]

  9. Hassold T.J., Sherman S.L., Pettay D., Page D.C., Jacobs P.A. XY chromosome nondisjunction in man is associated with diminished recombination in the pseudoautosomal region. Am. J. Hum. Genet. (1991) 49:253–260.[Web of Science][Medline]

  10. Savage A.R., Petersen M.B., Pettay D., Taft L., Allran K., Freeman S.B., Karadima G., Avramopoulos D., Torfs C., Mikkelsen M., et al. Elucidating the mechanisms of paternal non-disjunction of chromosome 21 in humans. Hum. Mol. Genet. (1998) 7:1221–1227.[Abstract/Free Full Text]

  11. Shi Q., Spriggs E., Field L., Ko E., Barclay L., Martin R. Single sperm typing demonstrates that reduced recombination is associated with the production of aneuploidy 24,XY human sperm. Am. J. Med. Genet. (2001) 99:34–38.[CrossRef][Web of Science][Medline]

  12. Hassold T., Merrill M., Adkins K., Freeman S., Sherman S. Recombination and maternal age-dependent non-disjunction: molecular studies of trisomy 16. Am. J. Hum. Genet. (1995) 57:867–874.[Web of Science][Medline]

  13. Lamb N.E., Freeman S.B., Savage-Austin A., Pettay D., Taft L., Hershey J., Gu Y., Shen J., Saker D., May K.M., et al. Susceptible chiasmate configurations of chromosome 21 predispose to non-disjunction in both maternal meiosis I and meiosis II. Nat. Genet. (1996) 14:400–405.[CrossRef][Web of Science][Medline]

  14. Baker S.M., Bronner C.E., Zhang L., Plug A.W., Robatzek M., Warren G., Elliott E.A., Yu J., Ashley T., Arnheim N., et al. Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell (1995) 82:309–319.[CrossRef][Web of Science][Medline]

  15. Edelmann W., Cohen P., Kane M., Lau K., Morrow B., Bennett S., Umar A., Kunkel T., Cattoretti G., Chaganti R., et al. Meiotic pachytene arrest in MLH1-deficient mice. Cell (1996) 85:1125–1134.[CrossRef][Web of Science][Medline]

  16. Yuan L., Liu J., Zhao J., Brundell E., Daneholt B., Hoog C. The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol. Cell. (2000) 5:73–83.[CrossRef][Web of Science][Medline]

  17. Barlow A.L., Hulten M.A. Crossing over analysis at pachytene in man. Eur. J. Hum. Genet. (1998) 6:350–358.[CrossRef][Web of Science][Medline]

  18. Lynn A., Koehler K.E., Judis L., Chan E.R., Cherry J.P., Schwartz S., Seftel A., Hunt P.A., Hassold T.J. Covariation of synaptonemal complex length and mammalian meiotic exchange rates. Science (2002) 296:2222–2225.[Abstract/Free Full Text]

  19. Gonsalves J., Sun F., Schlegel P.N., Turek P.J., Hopps C.V., Greene C., Martin R., Reijo Perra R.A. Defective recombination in infertile men. Hum. Mol. Genet. (2004) 13:2875–2883.[Abstract/Free Full Text]

  20. Codina-Pascual M., Oliver-Bonet M., Navarro J., Campillo M., Garcia F., Egozcue S., Abad C., Egozcue J., Benet J. Synapsis and meiotic recombination analyses: MLH1 focus in the XY as an indicator. Hum. Reprod. (2005) 8:2133–2139.

  21. Topping D., Brown P., Judis L., Schwartz S., Seftel A., Thomas A., Hassold T. Synaptic defects at meiosis I and non-obstructive azoospermia. Hum. Reprod. (2006) 21:3171–3177.[Abstract/Free Full Text]

  22. Sun F., Turek P., Greene C., Ko E., Rademaker A., Martin R.H. Abnormal progression through meiosis in men with nonobstructive azoospermia. Fertil. Steril. (2007) 87:565–571.[CrossRef][Web of Science][Medline]

  23. Ma S., Ferguson K.A., Arsovska S., Moens P., Chow V. Reduced recombination associated with the production of aneuploid sperm in an infertile man: a case report. Hum. Reprod. (2006) 21:980–985.[Abstract/Free Full Text]

  24. Hassold T., Judis L., Chan E.R., Schwartz S., Seftel A., Lynn A. Cytological studies of meiotic recombination in human males. Cytogenet. Genome Res. (2004) 107:249–255.[CrossRef][Web of Science][Medline]

  25. Sun F., Trpkov K., Rademaker A., Ko E., Martin R.H. Variation in meiotic recombination frequencies among human males. Hum. Genet. (2005) 116:172–178.[CrossRef][Web of Science][Medline]

  26. Roeder G.S., Bailis J.M. The pachytene checkpoint. Trends Genet. (2000) 16:395–403.[CrossRef][Web of Science][Medline]

  27. Hulten M., Eliasson R., Tillinger K.G. Low chiasma count and other meiotic irregularities in two infertile 46,XY men with spermatogenic arrest. Hereditas (1970) 65:285–290.[Web of Science][Medline]

  28. Micic M., Micic S., Diklic V. Low chiasma frequency as an aetiological factor in male infertility. Clin. Genet. (1982) 22:266–269.[Web of Science][Medline]

  29. Chaganti R.S.K., Jhanwar S.C., Ehrenbard L.T., Kourides I.A., Williams J.J. Genetically determined asynapsis, spermatogenic degeneration, and infertility in men. Am. J. Hum. Genet. (1980) 32:833–848.[Web of Science][Medline]

  30. Hale D.W. Is XY-recombination necessary for spermatocyte survival during mammalian spermatogenesis? Cytogenet. Cell. Genet. (1994) 65:278–282.[Web of Science][Medline]

  31. Baudat F., Manova K., Yuen J.P., Jasin M., Keeney S. Chromosome synapsis defects and sexually dimorphic meioitic progression in mice lacking Spo11. Mol. Cell. (2000) 6:989–998.[CrossRef][Web of Science][Medline]

  32. Liu L., Franco S., Spyropolous B., Moens P.B., Blasco M.A., Keefe D.L. Irregular telomeres impair meiotic synapsis and recombination in mice. Proc. Natl Acad. Sci. USA (2004) 101:6496–6501.[Abstract/Free Full Text]

  33. de Vries F.A., de Boer E., van den Bosch M., Baarends W.M., Ooms M., Yuan L., Liu J.G., van Zeeland A.A., Heyting C., Pastink A. Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation. Genes Dev. (2005) 19:1376–1389.[Abstract/Free Full Text]

  34. Baarends W.M., Wassenaar E., van der Laan R., Hoogerbrugge J., Sleddens-Linkels E., Hoeijmakers J.H., de Boer P., Grootegoed J.A. Silencing of unpaired chromatin and histone H2A ubiquination in mammalian meiosis. Mol. Cell. Biol. (2005) 25:1041–1053.[Abstract/Free Full Text]

  35. Turner J.M., Mahadevaiah S.K., Fernandez-Capetillo O., Nussenzweig A., Xu X., Deng C.X., Burgoyne P.S. Silencing of unsynapsed meiotic chromosomes in the mouse. Nat. Genet. (2005) 37:41–47.[Web of Science][Medline]

  36. Odorisio T., Rodriguez T.A., Evans E.P., Clarke A.R., Burgoyne P.S. The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-indendent apoptosis. Nat. Genet. (1998) 18:257–261.[CrossRef][Web of Science][Medline]

  37. Tease C., Hartshorne G., Hulten M. Altered patterns of meiotic recombination in human fetal oocytes with asynapsis and/or synaptonemal complex fragmentation at pachytene. Reprod. Biomed. Online (2006) 13:89–95.

  38. Geoffroy-Siraudin C., Aknin-Seiffer I., Metzler-Guillemain C., Ghalamoun-Slaimi R., Bonzi M.F., Levy R., Guichaoua M.R. Meiotic abnormalities in patients bearing complete AZFc deletion of Y chromosome. Hum. Reprod. (2007) 6:1567–1572.

  39. Tease C., Hulten M.A. Inter-sex variation in synaptonemal complex lengths largely determine the different recombination rates in male and female germ cells. Cytogenet. Genome. Res. (2004) 107:208–215.[CrossRef][Web of Science][Medline]

  40. Codina-Pascual M., Campillo M., Kraus J., Speicher M.R., Egozcue J., Navarro J., Benet J. Crossover frequency and synaptonemal complex length: their variability and effects on human male meiosis. Mol. Hum. Reprod. (2006) 12:123–133.[Abstract/Free Full Text]

  41. Sun F., Oliver-Bonet M., Liehr T., Starke J., Turek P., Ko E., Rademaker A., Martin R.H. Variation in MLH1 distribution in recombination maps for individual chromosomes form human males. Hum. Mol. Genet. (2006) 15:2376–2391.[Abstract/Free Full Text]

  42. Koehler K.E., Hawley R.S., Sherman S.L., Hassold T. Recombination and nondisjunciton in humans and flies. Hum. Mol. Genet. (1996) 5:1495–1504.[Abstract]

  43. Rubes J., Vozdova M., Oracova E., Perreault S.D. Individual variation in the frequency of sperm aneuploidy in humans. Cytogenet. Genome Res. (2005) 111:229–236.[CrossRef][Web of Science][Medline]

  44. Bernardini L., Gianaroli L., Fortini D., Conte N., Magli C., Cavani S., Gaggero G., Tindiglia C., Ragni N., Venturini P.L. Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients. Hum. Reprod. (2000) 15:2165–2172.[Abstract/Free Full Text]

  45. Martin R.H., Greene C., Rademaker A.W., Ko E., Chernos J. Analysis of aneuploidy in spermatozoa from testicular biopsies from men with nonobstructuve azoospermia. J. Androl. (2003) 24:100–103.[Abstract/Free Full Text]

  46. Rodrigo L., Rubio C., Mateu E., Simon C., Remoh J., Pelicer A., Gil-Salom M. Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by fluorescent in-situ hybridization. Hum. Reprod. (2004) 19:118–123.[Abstract/Free Full Text]

  47. Ma S., Arsovska S., Moens P., Nigro M., Chow V. Analysis of early meiotic events and sperm aneuploidy in non-obstructive azoospermic men: a preliminary report. Fertil. Steril. (2006) 85:646–652.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum ReprodHome page
K. A. Ferguson, S. Leung, D. Jiang, and S. Ma
Distribution of MLH1 foci and inter-focal distances in spermatocytes of infertile men
Hum. Reprod., June 1, 2009; 24(6): 1313 - 1321.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. H. Brown, I. R. Schultz, J. G. Cloud, and J. J. Nagler
Aneuploid sperm formation in rainbow trout exposed to the environmental estrogen 17{alpha}-ethynylestradiol
PNAS, December 16, 2008; 105(50): 19786 - 19791.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
G. Kirkpatrick, K. A. Ferguson, H. Gao, S. Tang, V. Chow, B. H. Yuen, and S. Ma
A comparison of sperm aneuploidy rates between infertile men with normal and abnormal karyotypes
Hum. Reprod., July 1, 2008; 23(7): 1679 - 1683.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R.H. Martin
Cytogenetic determinants of male fertility
Hum. Reprod. Update, June 4, 2008; (2008) dmn017v1.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/23/2870    most recent
ddm246v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ferguson, K. A.
Right arrow Articles by Ma, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ferguson, K. A.
Right arrow Articles by Ma, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?