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Joanna Gonsalves, Fei Sun, Peter N. Schlegel, Paul J. Turek, Carin V. Hopps, Calvin Greene, Renee H. Martin, Renee A. Reijo Pera, Defective recombination in infertile men, Human Molecular Genetics, Volume 13, Issue 22, 15 November 2004, Pages 2875–2883, https://doi.org/10.1093/hmg/ddh302
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Abstract
Two percent of men are infertile owing to defects in sperm production. In 10–15% of cases, Y chromosome deletions that encompass critical spermatogenesis genes are detected; in the remaining cases, the cause of infertility is unknown. In model organisms, defects in recombination genes cause infertility, germ cell aneuploidy and subsequent development of inviable or abnormal progeny. Several studies have also linked infertility and higher rates of germ cell aneuploidy in men and women. Thus, we reasoned that defective recombination may be a major cause of infertility in men with poor or no sperm production and we performed the first comparison of recombination parameters within populations of single spermatocytes from infertile and fertile men who reported for assisted reproduction. We observed that 10% of non-obstructive azoospermic men had significantly lower recombination frequencies than men with normal spermatogenesis. Furthermore, when we focused our analysis only on those men who had a pathological diagnosis of ‘maturation arrest’ due to arrest during sperm development, about half had detectable defects in recombination. In contrast, none of the men with normal spermatogenesis had defects in recombination. Thus, this study provides direct evidence that defects in recombination are linked to poor sperm production in a significant percentage of infertile men. Implications of this observation for the use of assisted reproductive technologies are especially relevant to consider, given that recombination is required to both introduce genetic variation and insure proper chromosome separation during meiosis.
INTRODUCTION
Infertility is a major health problem that affects ∼10–15% of couples; ∼40% of infertility cases are attributed to the male partner (1,2). Causes of male infertility can be divided into four general categories: defective sperm production, obstruction or physical blockage of the reproductive tract, inflammation or immunological dysfunction or sexual disorders such as impotence (3). The majority of cases with defective sperm production are idiopathic (3). In recent years, however, deletions of three regions on the Y chromosome have been linked to production of very few or no sperm; these regions of the Y chromosome are termed the AZF (azoospermia factor)-a, -b and -c regions (4–6). Apart from Y chromosome deletions, little is known about molecular causes or the defects in specific pathways that lead to spermatogenic disorders in men.
Historically, the lack of knowledge regarding male infertility has resulted in a few treatment options, but no other consequences, because men with very poor sperm production were unable to reproduce. In recent years, however, intracytoplasmic sperm injection (ICSI) has been used to help infertile men achieve biological paternity (7). In ICSI, a single sperm is injected directly into an oocyte to produce an embryo that is then transferred to the uterus after several days of development in culture (7). Normally, the production of an embryo results from the fusion of an oocyte with a single sperm, from a total population of the ∼200–300 million that are ejaculated (8). Given that ICSI requires that men produce just a single sperm, it is apparent that ICSI may skew the natural selection process and that one potential risk associated with its use is the transfer of genetic defects from one generation to the next. For example, some men with azoospermia (no sperm in semen) due to deletion in the AZFc region can father children through ICSI with rare sperm in their testis. However, their sons inherit the AZFc deletion and infertility (9). Because of this ability to pass genetic defects from one generation to the next, it is important to begin to understand the molecular causes of infertility in men.
In model organisms, well-defined meiotic checkpoints halt spermatogenesis in the face of defective recombination and lead to subsequent infertility (10–14). A few studies in the 1970s and 1980s demonstrated that some infertile men have testicular histology indicative of meiotic arrest and hypothesized that recombination errors may underlie male infertility (15–17). Technically, these studies were hampered in their ability to analyze the progression of meiosis, the precise stage of arrest, and the frequency and location of recombination sites because of limitations of existing techniques at that time to detect chromosomal chiasma. Recently, reagents that specifically mark sites of recombination have been used to examine the variation and fidelity of recombination in spermatocytes from fertile men (18,19). In these studies, the centromere of each chromosome in the spermatocyte was localized with CREST antisera and sites of meiotic recombination on the synaptonemal complex (SC) on the chromosome were visualized with antisera to DNA mismatch repair protein, MLH1 (Mut-L homolog 1) (19,20). Using these reagents, Lynn et al. (19) examined the frequency and location of recombination events within single spermatocytes of 14 individual men with normal spermatogenesis. They demonstrated that the overall mean number of MLH1 foci, which mark sites of genetic exchange, was 49.1±4.8 per germ cell with a range of 34–66 foci per germ cell, numbers that are remarkably similar to expectations based on human genetic maps constructed from Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees (19). Two recent case reports have also each identified a single individual who had meiotic defects characterized by inability to form the SC or establish recombination, by using these reagents (21,22).
In spite of these advances in analysis of recombination in men, there are no reports as to whether defective recombination is common and whether it occurs in men with diverse causes of infertility. Even more disquieting is the lack of information regarding outcomes of assisted reproduction and recombination parameters in infertility clinics. Thus, we used recently developed tools that allow the examination of up to several hundred cells per individual to directly determine and compare recombination frequencies in a population of 66 men characterized by their fertility status. In addition, because the infertile men presented to the clinic for assisted reproduction, we also determined whether deficiencies in recombination were compatible with sperm production, oocyte fertilization and embryo development.
RESULTS
Analysis of recombination in meiotic cells
Meiotic division begins with a period of cell division characterized by four distinct stages of prophase: the leptotene, zygotene, pachytene and diplotene stages. DNA replication begins in the pre-leptotene to leptotene stages and the sister chromatids begin to condense (Fig. 1A). Then in zygotene, the sister chromatids synapse along their length and form lateral elements that contain synaptonemal complex proteins (SCP), such as SCP2 and SCP3 (Fig. 1B and C). At pachytene, SC formation is complete and recombination nodules that contain proteins such as MLH1 are clearly visible (Fig. 1D and E). Finally, at the diplotene stage, homologous chromosomes begin to separate and only the chiasmata, the sites of the recombination machinery, hold the chromosomes together (Fig. 1F). Meiotic division continues to progress in an orderly fashion from meiosis I to meiosis II, unless errors in the recombination or chromosomal segregation machinery trigger arrest at one of two checkpoints, either during prophase or at the metaphase–anaphase transition (23,24). In men, completion of meiosis requires 30–35 days (25).
In this study, we analyzed testis tissue that was collected from 66 men in three different centers: New York, San Francisco and Alberta. The men in the study were classified into three different groups. Group I was the control group. This group contained 17 fertile men who had either had a previous vasectomy or had testicular cancer (or other types of cancer such as prostate cancer). All had histologically normal spermatogenesis. Groups II and III, together, contained 49 affected individuals who were azoospermic (they completely lacked sperm in their semen). These men underwent testicular biopsy in the hopes of finding sperm in their testis to be used for assisted reproduction. These groups differed from each other in that men in Group II were diagnosed with obstructive azoospermia (N=9). These men were infertile and were diagnosed with obstruction of the seminal tract, such as congenital bilateral absence of the vas deferens. All men in Group II had abundant sperm production in the testis. Because a molecular cause of obstruction is unlikely to be linked to recombination, we expected that even though these men were azoospermic, they should have normal recombination. Group III consisted of 40 men with non-obstructive azoospermia. Within this group, standard pathological diagnoses were used to categorize the testicular biopsies from these men as displaying ‘Sertoli cell only’ syndrome (SCO), hypospermatogenesis or maturation arrest (26). SCO syndrome is characterized by the complete absence of germ cells in the testis. Hypospermatogenesis is characterized by reduced spermatogenesis without a defined focal point of arrest. Maturation arrest is characterized by the cessation of germ cell development often at a specific stage, with or without the production of occasional mature spermatids. Histologically, men in Group III differed from men in Group II in that they produced very few or no sperm in the testis tissue, whereas spermatogenesis in men in Group II was histologically normal. No molecular causes of infertility in these men were known.
Arrest points in non-obstructive azoospermic men
To characterize defects in spermatogenesis, we first examined the progression of spermatogenesis using immunofluorescence markers of the SC and recombination machinery to determine the stage of spermatogenic arrest in non-obstructive infertile men. The progression of spermatogenesis in these men was then categorized according to whether we observed: (1) arrest of germ cell development prior to meiosis, (2) arrest of germ cell development at the zygotene stage of prophase I and a subsequent lack of germ cells beyond this stage or (3) the presence of mature meiotic cells (beyond the zygotene stage) in their testis. We found that 21 of 40 (or 53%) non-obstructive azoospermic men completely lacked meiotic germ cells. Just two of 40 (or 5%) non-obstructive azoospermic men had a complete arrest at the zygotene stage of prophase I [with no pachytene cells observed (Table 1)]. Another two of 40 (5%) had an incomplete arrest at zygotene with ∼2–3% of spermatogenic cells in the pachytene stage compared with 88% in the controls (Table 1). Finally, 15 of 40 men (or 38%) had mature meiotic cells indicative of progression beyond the zygotene stage of prophase I to pachytene.
Significantly lower levels of recombination and synapsis fidelity in some infertile men
The presence of pachytene cells in 15 infertile men with non-obstructive azoospermia allowed us to analyze their recombination frequency and synapsis fidelity compared with that of men in the control and obstructive azoospermic groups, using antisera that mark the centromere (CREST antisera), sites of meiotic recombination (MLH1 antisera) and SC (SCP3, SYN1) (Table 2; Fig. 2). We observed that our control group had an overall mean of 45.9±5.3 and a range of 42.5±3.9 to 50.4±6.2 mean MLH1 foci per pachytene spermatocyte (Table 2). The obstructive azoospermia group had an overall mean of 44.8±7.2 and a range of 42.0±3.9 to 50.9±4.8 mean MLH1 foci per pachytene spermatocyte (Table 2). These numbers closely resemble the recombination frequencies obtained in studies by Lynn et al. (19). The men in the group with idiopathic non-obstructive azoospermia had a lower mean of 40.0±12.2 and a range of just 4.2±2.4 to 48.9±7.4 mean MLH1 foci per pachytene spermatocyte (Table 2). We next sought to determine whether this recombination data from the different groups differed significantly. For this purpose, we used a non-parametric test because the data were not normally distributed. We noted that the mean number of recombination sites per pachytene cell between the control and obstructive azoospermic groups differed significantly (P<0.0009; Mann–Whitney test) as did both the control and the obstructive azoospermic groups when compared with the non-obstructive azoospermic groups (P<0.00001; Mann–Whitney test).
Next, to further focus on the differences between the groups, we compared the individual with the lowest mean in the control and obstructive groups with individuals in the non-obstructive azoospermic group. Comparisons of individuals across the groups indicated that four individual men diagnosed with maturation arrest in the group with non-obstructive azoospermia (10%) had a significantly lower mean number of MLH1 foci per pachytene cell when compared with all men in the control and obstructive azoospermia group (P<0.0001 for MA2, MA4, MA7, P<0.02 MA1; Mann–Whitney rank-sum test). We also noted that there was no significant difference between the individual with the lowest mean in the control group when compared with the individual with the lowest mean in the obstructive azoospermic group.
We next focused on the relationship between recombination parameters and pathological diagnoses of the 15 non-obstructive azoospermic men with pachytene cells present, in more detail. Of these 15 men, eight of them were diagnosed with hypospermatogenesis and seven were diagnosed with maturation arrest. The men with hypospermatogenesis had an overall mean of 45.0±7.0 MLH1 foci, whereas the men with maturation arrest had an overall mean of 34.0±14.6. We compared the men with hypospermatogenesis and maturation arrest with each other and with the control and obstructive azoospermic groups. We found that the mean number of recombination events per pachytene spermatocyte in men with hypospermatogenesis was significantly different (P<0.006; Mann–Whitney test) compared with the control group but not to the obstructive azoospermic group. The men with maturation arrest had a significantly different mean number of recombination events per pachytene spermatocyte compared with the control group, obstructive group and men with hypospermatogenesis (P<0.00001; Mann–Whitney test). Therefore, we concluded from this data that of men diagnosed with arrest during sperm maturation, fully four of seven (57%) were defective in recombination (Table 2).
In one center of our study (Alberta), the fidelity of chromosomal synapsis was also measured by tallying: (1) the frequency of incomplete synapsis of chromosomes within spermatocytes and (2) the frequency of cells with at least 1 bivalent with no MLH1 foci, (that is with no apparent crossovers on that bivalent) (Table 3; Fig. 3). The percentages of cells with incomplete synapsis and bivalents without MLH1 staining were then compared for the men in each group sampled in Alberta. We noted significant differences in fidelity of synapsis in the non-obstructive azoospermia group when compared with the control and obstructive azoospermia groups (P<0.00001 for control group, P<0.0006 for the obstructive group; Fisher exact test) (Table 3). There was no significant difference between the control and obstructive groups in fidelity of synapsis. There was a statistical difference in the number of cells with at least one bivalent without MLH1 foci between the control group and both obstructive and non-obstructive azoospermic groups (P<0.00001; Fisher exact test), but not between the obstructive and non-obstructive azoospermic groups. When we examined individuals, we found that one of four men from the group with non-obstructive azoospermia had a significantly higher percentage of cells with unpaired regions in the SC when compared with the control and obstructive azoospermia groups (P<0.00001; Fisher exact test). Moreover, two of four men from the group with non-obstructive azoospermia had a significantly higher percentage of cells with at least 1 bivalent with no MLH1 foci when compared with the control group (P<0.008; Fisher exact test). One of these men also demonstrated a significant difference (P<0.00001; Fisher exact test) when compared with men in the obstructive azoospermia group.
Defective recombination and/or a lower fidelity of synapsis and assisted reproduction
Given the defects in recombination that we observed, we examined the outcomes of assisted reproduction in men in our study. In particular, examination of clinical outcomes indicated that of the four men who had an overall reduced recombination frequency, only one man (MA2) produced sperm that was used in assisted reproduction (Fig. 4). Curiously, this man had the most severely affected recombination parameters that we observed; he had the lowest recombination frequency with a mean of just 4.2±2.4 MLH1 foci per pachytene spermatocyte. When sperm from MA2 was used to fertilize six oocytes, fertilization occurred and embryos were subsequently transferred to the uterus. However, there was no pregnancy after transfer.
DISCUSSION
When we classified germ cells according to their stage of development in infertile men with non-obstructive azoospermia, we found that 21 out of 40 (53%) non-obstructive azoospermic men lacked meiotic germ cells. These men were diagnosed with SCO syndrome, which is characterized by the absence of germ cells in the testis. Another four of 40 (10%) non-obstructive azoospermic men had germ cells arrested completely or nearly completely at the zygotene stage of prophase in meiosis I. This stage of germ cell arrest, and subsequent infertility, is identical to that observed in mice with mutations in recombination genes such as Spo11, Dmc1, Msh4 and Msh5 (10–14). In these mutant mice, germ cells arrest at zygotene in meiosis I and are subsequently eliminated via apoptosis. Thus, there is a checkpoint or genetic safeguard that eliminates products of faulty meiotic recombination at this stage of spermatogenesis in mice (27).
The remaining 15 of 40 men progressed through any zygotene checkpoints that might have been imposed. We compared the recombination frequency of these men with non-obstructive azoospermia with that of control men and men with obstructive azoospermia. Men in the control group demonstrated recombination frequencies that were similar to those in previous studies (18,19). In contrast, we were surprised to observe that the recombination frequency in men with obstructive azoospermia was significantly lower than that of the control men. Men with obstructive azoospermia generally are diagnosed with physical causes of azoospermia such as blockage of spermatogenic ducts or absence of the vas deferens. Although the difference in recombination frequency between these men and controls was small, it was significant. Notably, it has long been observed that physical factors such as increased temperature, varicoceles (a physical blockage) or a history of undescended testis lead to decreased fertility. It is interesting to speculate that these physical disturbances might affect a slight reduction in recombination, as in men with obstructive azoospermia. Alternatively, the decreased recombination may be due to microenvironmental effects or spermatogenic disorders that coexist with obstruction in the testis.
We observed the largest and most significant difference in mean number of recombination foci per pachytene spermatocyte in the non-obstructive azoospermic group when compared with both the control and obstructive azoospermic groups. When we compared recombination frequencies of men with hypospermatogenesis, or reduced spermatogenesis, we found that it differed significantly from that of men in the control group but not the obstructive azoospermic group. We do not, however, understand the molecular basis for this difference at this time; nonetheless, the causes may parallel those that underlie the effects observed in the obstructive azoospermic group, in that although spermatogenesis is completed, it is done so with aberrant parameters due to microenvironmental or coexisting conditions. Finally, we noted that men who were pathologically diagnosed with ‘maturation arrest’ during germ cell differentiation had significantly different recombination frequencies from those in the control group, obstructive group or other non-obstructive azoospermic men diagnosed with hypospermatogenesis. Four men diagnosed a priori with maturation arrest had significantly lower recombination frequencies than men with normal spermatogenesis or men with hypospermatogenesis. Therefore, of those diagnosed with germ cell maturation arrest, 57%, or four of seven, were defective in recombination. Although there is a clear variation in recombination frequencies within and among fertile individuals, we observed that these four non-obstructive azoospermic individuals had significantly lower recombination frequencies compared with all men in either the control and obstructive azoospermic groups. Whereas two of the individuals had what might be considered modest differences in recombination parameters, the other two were well outside the range of recombination seen in men with normal spermatogenesis.
Deletions of genes on the Y chromosome, the most common genetically defined cause of infertility in men, account for ∼10–15% of severe spermatogenic disorders (28). Thus, the observation here that 10% of non-obstructive azoospermic men had significantly reduced recombination frequencies in our study represents a frequent correlate of infertility. Defects in recombination, uncovered here, would constitute the second most common defined cause of infertility, which is also likely to be genetically based.
Finally, we noted in this study that the individual with the most severe defect in recombination produced a few rare sperm for use in ICSI. Thus, clearly and contrary to common belief, severe defects in recombination in men cannot be incompatible with sperm production; moreover, sperm from men with defective recombination are likely to be used routinely, and without knowledge of recombination status, in assisted reproductive clinics. Taken together, our observations indicate that azoospermic men, in particular those diagnosed with maturation arrest, are at a great risk of carrying defects in recombination. Because decreased recombination has been linked to chromosome abnormalities in model organisms and humans, our results suggest that these infertile men may be at a higher risk of producing chromosomally abnormal offspring (29–31). Therefore, we would suggest that assessment of recombination parameters in even larger populations of infertile men is warranted in order to determine the spectrum of outcomes associated with the use of sperm from men with defective recombination for embryo production, in order to provide appropriate counsel regarding risks, and in order to set the stage for gene mapping studies in the infertile population.
MATERIALS AND METHODS
Tissue collection
Informed consent for collection of testis tissues was obtained from patients presenting for treatment of male infertility, reverse vasectomies or testicular cancer at Cornell University Medical Center (Institutional Review Board), University of California at San Francisco (Committee on Human Research) and the University of Calgary (Conjoint Health Research Ethics Board), as appropriate. All of the procedures were in accordance with the responsible committee on human experiments and with the Helsinki Declaration of 1975, as revised in 1983. In most cases, one-third of the tissue sample was processed immediately for meiotic analysis, another third was preserved individually at −80°C for molecular analysis and the remainder was fixed in Bouin's fixative for histological analysis.
Meiotic analysis
Testis tissue was processed as previously reported (18,19). Primary antibodies used at the University of Calgary were human anti-CREST (gift from M. Fritzler, University of Calgary) rabbit anti-MLH1 (Oncogene), goat anti-SCP3 (gift from T. Ashley, Yale University) and mouse anti-SYN1 (gift from P. Moens, York University). Primary antibodies used at Cornell University and the University of California, San Francisco (UCSF) were human anti-CREST (gift from Bill Brinkley, Baylor College of Medicine), rabbit or mouse anti-MLH1 (Oncogene) and goat anti-SCP3 (gift from T. Ashley, Yale University) or rabbit anti-rat SCP3 (gift from Christa Heyting, Wageningen University). For secondary antibodies, AMCA donkey anti-human (Jackson ImmunoResearch), Alexa 488 donkey anti-rabbit (Molecular Probes), Alexa 55 donkey anti-goat (Molecular Probes) and Cy3 donkey anti-mouse (Jackson ImmunoResearch) were used at the University of Calgary. AMCA donkey anti-human (Jackson ImmunoResearch), FITC donkey anti-rabbit (Jackson ImmunoResearch) and Rd donkey anti-goat or anti-mouse (Jackson ImmunoResearch) were used at Cornell and UCSF. Slides were scanned with a Fluorescent Leica DMRB microscope and images of SCs, MLH1 and CREST locations were captured on a Leica DFC 300F camera. Prints of captured images were analyzed in order to determine the number of MLH1 foci on each individual SC and in the whole cell. We attempted to analyze 100 pachytene spermatocytes per man whenever possible. However, in some cases, men produced too few spermatocytes to reach this goal and we analyzed fewer cells in those cases. Note that all scoring was done blindly, in respect to infertility diagnosis, and that a subset of samples were scored independently by two scientists (three of 40).
Statistical analysis
The statistical package Statistics/Data Analysis (STATA) was used for statistical analysis. We first tested whether there was a statistical difference in the mean number of MLH1 foci per pachytene spermatocyte between men from the three centers in the control group using the Mann–Whitney test. We found that there were no statistical differences.
Next, to determine whether the mean number of MLH1 foci differed between the control, obstructive and non-obstructive groups, we used a non-parametric statistical method, the Mann–Whitney test. This test was chosen because the MLH1 parameters were not normally distributed. The non-normal distribution was apparent in examining the values in Table 2. When plotting the mean MLH1 foci per cell in each individual it became apparent that they were skewed. Some examples of men with skewed distributions are O6, O7, HY5 and MA2. With the data that are not normally distributed, non-parametric methods are recommended and more reliable than parametric methods. When the t-test is used on data that are not normally distributed, the significance probabilities can be changed and the sensitivity or power of the test in finding a significant result, when the null hypothesis is false, is altered. Thus, we chose the Mann–Whitney test as a more stringent measure of significance than the parametric t-test. We used this test to compare the mean between the control, obstructive and non-obstructive azoospermic groups. Because of the variability between and among individuals we could not just compare the groups. To narrow in on the differences between the groups, we also used the Mann–Whitney test to compare the individual with the lowest mean MLH1 foci in the control and obstructive azoospermia groups with each other and with each individual in the non-obstructive group; finally we used the Mann–Whitney test to compare the men with hypospermatogenesis and maturation arrest in the non-obstructive azoospermic group with each other and with the control and obstructive azoospermic groups.
To determine whether the fidelity of chromosome synapsis differed between groups, we used the Fisher exact test. The Fisher exact test is frequently recommended in small studies when the expected frequency is smaller than 5. This test is used to determine if there are non-random associations between two categorical variables, similar to the χ2 test. Using the Fisher exact test, we compared the percentage of cells with incomplete synapsis and percentage of cells with at least one bivalent without any MLH1 foci between the control, obstructive and non-obstructive azoospermic groups. We also compared the individuals in the control and obstructive azoospermia groups who had the highest percentages of cells with incomplete synapsis and the highest percentage of cells with at least 1 bivalent without MLH1 foci to each individual in the non-obstructive azoospermia group via the Fisher exact test.
ACKNOWLEDGEMENTS
We thank T. Ashley, B. Brinkley, M. Fritzler, C. Heyting and P. Moens for the generous gift of antibodies, M. Abeyta, A. Clark, L. Judis, E. Ko and M. Phillips for technical assistance, Terry Hassold for guidance in analysis of synaptonemal complexes, D. Hardy, M. Hardy and A. Mielnik for their assistance at the Population Council in New York, K. Trpkov and G. Kozak for patient samples that were obtained in Calgary and A. Clark, U. Ezeh, R. Taylor, J. Tung, R. Weiner and E. Xu at UCSF for critical review of the manuscript. This work was supported by the Canadian Institutes of Health Research (MA7961 to R.H.M. and a fellowship to F.S.); by the Canada Research Chair in Genetics (R.H.M.), a fellowship from the National Institute of General Medical Sciences (1RS25GM56847 to J.G.) and the National Institute of Child Health and Human Development (RO1HD38987) and the Sandler Family Foundation to R.A.R.P.
Patient . | Age (years) . | No. of cells analyzed . | Percentage of cell types . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|
MA8 | 33 | 42 | 26% L, 3% P, 71% Z | No | No | No |
MA9 | 49 | 55 | 33% L, 2% P, 65% Z | Yes | Yes | Yes |
MA10 | 41 | 100 | 13% L, 0% P, 87% Z | No | No | No |
MA11 | 35 | 100 | 28% L, 0% P, 72% Z | No | No | No |
Controls | 7% L, 88% P, 4% Z |
Patient . | Age (years) . | No. of cells analyzed . | Percentage of cell types . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|
MA8 | 33 | 42 | 26% L, 3% P, 71% Z | No | No | No |
MA9 | 49 | 55 | 33% L, 2% P, 65% Z | Yes | Yes | Yes |
MA10 | 41 | 100 | 13% L, 0% P, 87% Z | No | No | No |
MA11 | 35 | 100 | 28% L, 0% P, 72% Z | No | No | No |
Controls | 7% L, 88% P, 4% Z |
Abbreviations: MA, maturation arrest; P, pachytene; L, leptotene; Z, zygotene.
Patient . | Age (years) . | No. of cells analyzed . | Percentage of cell types . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|
MA8 | 33 | 42 | 26% L, 3% P, 71% Z | No | No | No |
MA9 | 49 | 55 | 33% L, 2% P, 65% Z | Yes | Yes | Yes |
MA10 | 41 | 100 | 13% L, 0% P, 87% Z | No | No | No |
MA11 | 35 | 100 | 28% L, 0% P, 72% Z | No | No | No |
Controls | 7% L, 88% P, 4% Z |
Patient . | Age (years) . | No. of cells analyzed . | Percentage of cell types . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|
MA8 | 33 | 42 | 26% L, 3% P, 71% Z | No | No | No |
MA9 | 49 | 55 | 33% L, 2% P, 65% Z | Yes | Yes | Yes |
MA10 | 41 | 100 | 13% L, 0% P, 87% Z | No | No | No |
MA11 | 35 | 100 | 28% L, 0% P, 72% Z | No | No | No |
Controls | 7% L, 88% P, 4% Z |
Abbreviations: MA, maturation arrest; P, pachytene; L, leptotene; Z, zygotene.
Patient . | Age . | No. of cells analyzed . | Mean no. of MLH1 foci (±SD) . | Range . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|---|
Controls (N=17) | |||||||
C1 | 49 | 40 | 43.5±3.8 | 37–54 | Yes | N/A | — |
C2 | 57 | 67 | 44.8±3.9 | 34–58 | Yes | N/A | — |
C3 | 42 | 26 | 49.0±6.4 | 35–61 | Yes | N/A | — |
C4 | 53 | 70 | 46.8±4.9 | 35–59 | N/A | N/A | — |
C5 | 44 | 48 | 47.8±4.5 | 38–56 | N/A | N/A | — |
C6 | 54 | 47 | 48.0±3.9 | 40–57 | N/A | N/A | — |
C7 | 51 | 62 | 45.6±4.2 | 36–55 | Yes | N/A | — |
C8 | 56 | 94 | 44.4±4.7 | 32–53 | Yes | N/A | — |
C9 | 46 | 46 | 42.6±5.9 | 26–51 | N/A | N/A | — |
C10 | 40 | 52 | 50.4±6.2 | 39–66 | Yes | N/A | — |
C11 | 48 | 79 | 43.4±4.1 | 35–53 | Yes | N/A | — |
C12 | 47 | 100 | 49.9±4.3 | 38–62 | N/A | N/A | — |
C13 | 22 | 100 | 45.3±5.6 | 22–60 | N/A | N/A | — |
C14 | 26 | 100 | 42.5±3.9 | 32–52 | N/A | N/A | — |
C15 | 81 | 100 | 46.4±5.0 | 25–58 | N/A | N/A | — |
C16 | 79 | 100 | 46.6±5.9 | 21–58 | N/A | N/A | — |
C17 | 80 | 100 | 45.8±4.1 | 34–57 | N/A | N/A | — |
Obstructive azoospermia (N=9) | |||||||
O1 | 31 | 18 | 44.8±8.8 | 14–56 | Yes | Yes | Yes |
O2 | 44 | 23 | 50.9±4.8 | 40–62 | Yes | Yes | No |
O3 | 32 | 62 | 47.2±5.1 | 35–57 | Yes | Yes | Yes |
O4 | 38 | 95 | 44.2±5.1 | 31–57 | Yes | Yes | Yes |
O5 | 29 | 66 | 42.0±3.9 | 32–51 | Yes | N/A | — |
O6 | 36 | 100 | 44.9±9.9 | 4–60 | Yes | Yes | No |
O7 | 29 | 100 | 43.0±8.8 | 10–58 | Yes | Yes | Yes |
O8 | 31 | 34 | 50.4±6.3 | 29–61 | Yes | Yes | Yes |
O9 | 34 | 99 | 44.1±4.6 | 29–54 | Yes | Yes | No |
Non-obstructive azoospermia (idiopathic) (N=15) | |||||||
HY1 | 40 | 88 | 45.3±5.3 | 20–54 | Yes | Yes | Yes |
HY2 | 24 | 100 | 48.9±7.4 | 23–61 | Yes | No | No |
HY3 | 43 | 74 | 43.7±7.8 | 8–56 | Yes | Yes | Yes |
HY4 | 47 | 48 | 42.2±6.7 | 18–54 | Yes | Yes | No |
HY5 | 54 | 28 | 42.7±6.6 | 24–51 | Yes | No | No |
HY6 | 49 | 64 | 42.7±3.1 | 35–48 | Yes | N/A | — |
HY7 | 42 | 78 | 46.1±3.4 | 38–55 | Yes | Yes | Yes |
HY8 | 34 | 100 | 42.7±9.0 | 15–61 | Yes | Yes | Yes |
MA1 | 34 | 22 | 32.7±17.4 | 1–60 | No | No | No |
MA2 | 31 | 60 | 4.2±2.4 | 1–11 | Yes | Yes | No |
MA3 | 25 | 71 | 44.3±4.5 | 31–52 | No | No | No |
MA4 | 37 | 48 | 23.5±7.5 | 8–40 | No | No | No |
MA5 | 27 | 53 | 41.0±6.1 | 17–55 | No | No | No |
MA6 | 33 | 100 | 41.5±5.5 | 3–52 | Yes | Yes | No |
MA7 | 35 | 100 | 37.9±7.0 | 16–52 | No | No | No |
SCO: no pachytene cells (N=21) | |||||||
Arrest at zygotene (N=4) | |||||||
Total=66 |
Patient . | Age . | No. of cells analyzed . | Mean no. of MLH1 foci (±SD) . | Range . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|---|
Controls (N=17) | |||||||
C1 | 49 | 40 | 43.5±3.8 | 37–54 | Yes | N/A | — |
C2 | 57 | 67 | 44.8±3.9 | 34–58 | Yes | N/A | — |
C3 | 42 | 26 | 49.0±6.4 | 35–61 | Yes | N/A | — |
C4 | 53 | 70 | 46.8±4.9 | 35–59 | N/A | N/A | — |
C5 | 44 | 48 | 47.8±4.5 | 38–56 | N/A | N/A | — |
C6 | 54 | 47 | 48.0±3.9 | 40–57 | N/A | N/A | — |
C7 | 51 | 62 | 45.6±4.2 | 36–55 | Yes | N/A | — |
C8 | 56 | 94 | 44.4±4.7 | 32–53 | Yes | N/A | — |
C9 | 46 | 46 | 42.6±5.9 | 26–51 | N/A | N/A | — |
C10 | 40 | 52 | 50.4±6.2 | 39–66 | Yes | N/A | — |
C11 | 48 | 79 | 43.4±4.1 | 35–53 | Yes | N/A | — |
C12 | 47 | 100 | 49.9±4.3 | 38–62 | N/A | N/A | — |
C13 | 22 | 100 | 45.3±5.6 | 22–60 | N/A | N/A | — |
C14 | 26 | 100 | 42.5±3.9 | 32–52 | N/A | N/A | — |
C15 | 81 | 100 | 46.4±5.0 | 25–58 | N/A | N/A | — |
C16 | 79 | 100 | 46.6±5.9 | 21–58 | N/A | N/A | — |
C17 | 80 | 100 | 45.8±4.1 | 34–57 | N/A | N/A | — |
Obstructive azoospermia (N=9) | |||||||
O1 | 31 | 18 | 44.8±8.8 | 14–56 | Yes | Yes | Yes |
O2 | 44 | 23 | 50.9±4.8 | 40–62 | Yes | Yes | No |
O3 | 32 | 62 | 47.2±5.1 | 35–57 | Yes | Yes | Yes |
O4 | 38 | 95 | 44.2±5.1 | 31–57 | Yes | Yes | Yes |
O5 | 29 | 66 | 42.0±3.9 | 32–51 | Yes | N/A | — |
O6 | 36 | 100 | 44.9±9.9 | 4–60 | Yes | Yes | No |
O7 | 29 | 100 | 43.0±8.8 | 10–58 | Yes | Yes | Yes |
O8 | 31 | 34 | 50.4±6.3 | 29–61 | Yes | Yes | Yes |
O9 | 34 | 99 | 44.1±4.6 | 29–54 | Yes | Yes | No |
Non-obstructive azoospermia (idiopathic) (N=15) | |||||||
HY1 | 40 | 88 | 45.3±5.3 | 20–54 | Yes | Yes | Yes |
HY2 | 24 | 100 | 48.9±7.4 | 23–61 | Yes | No | No |
HY3 | 43 | 74 | 43.7±7.8 | 8–56 | Yes | Yes | Yes |
HY4 | 47 | 48 | 42.2±6.7 | 18–54 | Yes | Yes | No |
HY5 | 54 | 28 | 42.7±6.6 | 24–51 | Yes | No | No |
HY6 | 49 | 64 | 42.7±3.1 | 35–48 | Yes | N/A | — |
HY7 | 42 | 78 | 46.1±3.4 | 38–55 | Yes | Yes | Yes |
HY8 | 34 | 100 | 42.7±9.0 | 15–61 | Yes | Yes | Yes |
MA1 | 34 | 22 | 32.7±17.4 | 1–60 | No | No | No |
MA2 | 31 | 60 | 4.2±2.4 | 1–11 | Yes | Yes | No |
MA3 | 25 | 71 | 44.3±4.5 | 31–52 | No | No | No |
MA4 | 37 | 48 | 23.5±7.5 | 8–40 | No | No | No |
MA5 | 27 | 53 | 41.0±6.1 | 17–55 | No | No | No |
MA6 | 33 | 100 | 41.5±5.5 | 3–52 | Yes | Yes | No |
MA7 | 35 | 100 | 37.9±7.0 | 16–52 | No | No | No |
SCO: no pachytene cells (N=21) | |||||||
Arrest at zygotene (N=4) | |||||||
Total=66 |
Abbreviations: C, control; O, obstructive azoospermia; HY, hypospermatogenesis; MA, maturation arrest; N/A, not available; SCO, Sertoli cell only syndrome. Italicized entries indicate men with significant reductions in recombination, as noted by reduced number of MLH1 foci per cell.
Patient . | Age . | No. of cells analyzed . | Mean no. of MLH1 foci (±SD) . | Range . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|---|
Controls (N=17) | |||||||
C1 | 49 | 40 | 43.5±3.8 | 37–54 | Yes | N/A | — |
C2 | 57 | 67 | 44.8±3.9 | 34–58 | Yes | N/A | — |
C3 | 42 | 26 | 49.0±6.4 | 35–61 | Yes | N/A | — |
C4 | 53 | 70 | 46.8±4.9 | 35–59 | N/A | N/A | — |
C5 | 44 | 48 | 47.8±4.5 | 38–56 | N/A | N/A | — |
C6 | 54 | 47 | 48.0±3.9 | 40–57 | N/A | N/A | — |
C7 | 51 | 62 | 45.6±4.2 | 36–55 | Yes | N/A | — |
C8 | 56 | 94 | 44.4±4.7 | 32–53 | Yes | N/A | — |
C9 | 46 | 46 | 42.6±5.9 | 26–51 | N/A | N/A | — |
C10 | 40 | 52 | 50.4±6.2 | 39–66 | Yes | N/A | — |
C11 | 48 | 79 | 43.4±4.1 | 35–53 | Yes | N/A | — |
C12 | 47 | 100 | 49.9±4.3 | 38–62 | N/A | N/A | — |
C13 | 22 | 100 | 45.3±5.6 | 22–60 | N/A | N/A | — |
C14 | 26 | 100 | 42.5±3.9 | 32–52 | N/A | N/A | — |
C15 | 81 | 100 | 46.4±5.0 | 25–58 | N/A | N/A | — |
C16 | 79 | 100 | 46.6±5.9 | 21–58 | N/A | N/A | — |
C17 | 80 | 100 | 45.8±4.1 | 34–57 | N/A | N/A | — |
Obstructive azoospermia (N=9) | |||||||
O1 | 31 | 18 | 44.8±8.8 | 14–56 | Yes | Yes | Yes |
O2 | 44 | 23 | 50.9±4.8 | 40–62 | Yes | Yes | No |
O3 | 32 | 62 | 47.2±5.1 | 35–57 | Yes | Yes | Yes |
O4 | 38 | 95 | 44.2±5.1 | 31–57 | Yes | Yes | Yes |
O5 | 29 | 66 | 42.0±3.9 | 32–51 | Yes | N/A | — |
O6 | 36 | 100 | 44.9±9.9 | 4–60 | Yes | Yes | No |
O7 | 29 | 100 | 43.0±8.8 | 10–58 | Yes | Yes | Yes |
O8 | 31 | 34 | 50.4±6.3 | 29–61 | Yes | Yes | Yes |
O9 | 34 | 99 | 44.1±4.6 | 29–54 | Yes | Yes | No |
Non-obstructive azoospermia (idiopathic) (N=15) | |||||||
HY1 | 40 | 88 | 45.3±5.3 | 20–54 | Yes | Yes | Yes |
HY2 | 24 | 100 | 48.9±7.4 | 23–61 | Yes | No | No |
HY3 | 43 | 74 | 43.7±7.8 | 8–56 | Yes | Yes | Yes |
HY4 | 47 | 48 | 42.2±6.7 | 18–54 | Yes | Yes | No |
HY5 | 54 | 28 | 42.7±6.6 | 24–51 | Yes | No | No |
HY6 | 49 | 64 | 42.7±3.1 | 35–48 | Yes | N/A | — |
HY7 | 42 | 78 | 46.1±3.4 | 38–55 | Yes | Yes | Yes |
HY8 | 34 | 100 | 42.7±9.0 | 15–61 | Yes | Yes | Yes |
MA1 | 34 | 22 | 32.7±17.4 | 1–60 | No | No | No |
MA2 | 31 | 60 | 4.2±2.4 | 1–11 | Yes | Yes | No |
MA3 | 25 | 71 | 44.3±4.5 | 31–52 | No | No | No |
MA4 | 37 | 48 | 23.5±7.5 | 8–40 | No | No | No |
MA5 | 27 | 53 | 41.0±6.1 | 17–55 | No | No | No |
MA6 | 33 | 100 | 41.5±5.5 | 3–52 | Yes | Yes | No |
MA7 | 35 | 100 | 37.9±7.0 | 16–52 | No | No | No |
SCO: no pachytene cells (N=21) | |||||||
Arrest at zygotene (N=4) | |||||||
Total=66 |
Patient . | Age . | No. of cells analyzed . | Mean no. of MLH1 foci (±SD) . | Range . | Sperm found . | Embryos after ICSI . | Confirmed pregnancy . |
---|---|---|---|---|---|---|---|
Controls (N=17) | |||||||
C1 | 49 | 40 | 43.5±3.8 | 37–54 | Yes | N/A | — |
C2 | 57 | 67 | 44.8±3.9 | 34–58 | Yes | N/A | — |
C3 | 42 | 26 | 49.0±6.4 | 35–61 | Yes | N/A | — |
C4 | 53 | 70 | 46.8±4.9 | 35–59 | N/A | N/A | — |
C5 | 44 | 48 | 47.8±4.5 | 38–56 | N/A | N/A | — |
C6 | 54 | 47 | 48.0±3.9 | 40–57 | N/A | N/A | — |
C7 | 51 | 62 | 45.6±4.2 | 36–55 | Yes | N/A | — |
C8 | 56 | 94 | 44.4±4.7 | 32–53 | Yes | N/A | — |
C9 | 46 | 46 | 42.6±5.9 | 26–51 | N/A | N/A | — |
C10 | 40 | 52 | 50.4±6.2 | 39–66 | Yes | N/A | — |
C11 | 48 | 79 | 43.4±4.1 | 35–53 | Yes | N/A | — |
C12 | 47 | 100 | 49.9±4.3 | 38–62 | N/A | N/A | — |
C13 | 22 | 100 | 45.3±5.6 | 22–60 | N/A | N/A | — |
C14 | 26 | 100 | 42.5±3.9 | 32–52 | N/A | N/A | — |
C15 | 81 | 100 | 46.4±5.0 | 25–58 | N/A | N/A | — |
C16 | 79 | 100 | 46.6±5.9 | 21–58 | N/A | N/A | — |
C17 | 80 | 100 | 45.8±4.1 | 34–57 | N/A | N/A | — |
Obstructive azoospermia (N=9) | |||||||
O1 | 31 | 18 | 44.8±8.8 | 14–56 | Yes | Yes | Yes |
O2 | 44 | 23 | 50.9±4.8 | 40–62 | Yes | Yes | No |
O3 | 32 | 62 | 47.2±5.1 | 35–57 | Yes | Yes | Yes |
O4 | 38 | 95 | 44.2±5.1 | 31–57 | Yes | Yes | Yes |
O5 | 29 | 66 | 42.0±3.9 | 32–51 | Yes | N/A | — |
O6 | 36 | 100 | 44.9±9.9 | 4–60 | Yes | Yes | No |
O7 | 29 | 100 | 43.0±8.8 | 10–58 | Yes | Yes | Yes |
O8 | 31 | 34 | 50.4±6.3 | 29–61 | Yes | Yes | Yes |
O9 | 34 | 99 | 44.1±4.6 | 29–54 | Yes | Yes | No |
Non-obstructive azoospermia (idiopathic) (N=15) | |||||||
HY1 | 40 | 88 | 45.3±5.3 | 20–54 | Yes | Yes | Yes |
HY2 | 24 | 100 | 48.9±7.4 | 23–61 | Yes | No | No |
HY3 | 43 | 74 | 43.7±7.8 | 8–56 | Yes | Yes | Yes |
HY4 | 47 | 48 | 42.2±6.7 | 18–54 | Yes | Yes | No |
HY5 | 54 | 28 | 42.7±6.6 | 24–51 | Yes | No | No |
HY6 | 49 | 64 | 42.7±3.1 | 35–48 | Yes | N/A | — |
HY7 | 42 | 78 | 46.1±3.4 | 38–55 | Yes | Yes | Yes |
HY8 | 34 | 100 | 42.7±9.0 | 15–61 | Yes | Yes | Yes |
MA1 | 34 | 22 | 32.7±17.4 | 1–60 | No | No | No |
MA2 | 31 | 60 | 4.2±2.4 | 1–11 | Yes | Yes | No |
MA3 | 25 | 71 | 44.3±4.5 | 31–52 | No | No | No |
MA4 | 37 | 48 | 23.5±7.5 | 8–40 | No | No | No |
MA5 | 27 | 53 | 41.0±6.1 | 17–55 | No | No | No |
MA6 | 33 | 100 | 41.5±5.5 | 3–52 | Yes | Yes | No |
MA7 | 35 | 100 | 37.9±7.0 | 16–52 | No | No | No |
SCO: no pachytene cells (N=21) | |||||||
Arrest at zygotene (N=4) | |||||||
Total=66 |
Abbreviations: C, control; O, obstructive azoospermia; HY, hypospermatogenesis; MA, maturation arrest; N/A, not available; SCO, Sertoli cell only syndrome. Italicized entries indicate men with significant reductions in recombination, as noted by reduced number of MLH1 foci per cell.
Patient . | Age (years) . | No. of pachytene cells analyzed . | Percentage of cells with incomplete synapsis . | Percentage of cells with at least 1 bivalent with 0 MLH1 . |
---|---|---|---|---|
Controls | ||||
C12 | 47 | 100 | 0 | 0 |
C13 | 22 | 100 | 0 | 5 |
C14 | 26 | 100 | 1 | 0 |
C15 | 81 | 100 | 9 | 11 |
C16 | 79 | 100 | 6 | 2 |
C17 | 80 | 100 | 0 | 8 |
Overall | 3 | 4 | ||
Obstructive azoospermia | ||||
O6 | 36 | 100 | 5 | 10 |
O7 | 29 | 100 | 2 | 22 |
Overall | 4 | 16 | ||
Non-obstructive azoospermia | ||||
HY1 | 40 | 88 | 6 | 26 |
HY2 | 24 | 100 | 8 | 7 |
HY8 | 34 | 100 | 10 | 16 |
MA1a | 34 | 22 | 63.6 | 72.7 |
Overall | 12 | 20 |
Patient . | Age (years) . | No. of pachytene cells analyzed . | Percentage of cells with incomplete synapsis . | Percentage of cells with at least 1 bivalent with 0 MLH1 . |
---|---|---|---|---|
Controls | ||||
C12 | 47 | 100 | 0 | 0 |
C13 | 22 | 100 | 0 | 5 |
C14 | 26 | 100 | 1 | 0 |
C15 | 81 | 100 | 9 | 11 |
C16 | 79 | 100 | 6 | 2 |
C17 | 80 | 100 | 0 | 8 |
Overall | 3 | 4 | ||
Obstructive azoospermia | ||||
O6 | 36 | 100 | 5 | 10 |
O7 | 29 | 100 | 2 | 22 |
Overall | 4 | 16 | ||
Non-obstructive azoospermia | ||||
HY1 | 40 | 88 | 6 | 26 |
HY2 | 24 | 100 | 8 | 7 |
HY8 | 34 | 100 | 10 | 16 |
MA1a | 34 | 22 | 63.6 | 72.7 |
Overall | 12 | 20 |
aSignificantly higher percentage of incomplete synapsis and significantly higher percentage of cells with at least one bivalent without MLH1 foci when compared with both groups.
Abbreviations: C, control; O, obstructive azoospermia; HY, hypospermatogenesis; MA, maturation arrest. Italicized entries point out men who had a significantly higher percentage of cells with at least 1 bivalent without MLH1 foci compared with the control group.
Patient . | Age (years) . | No. of pachytene cells analyzed . | Percentage of cells with incomplete synapsis . | Percentage of cells with at least 1 bivalent with 0 MLH1 . |
---|---|---|---|---|
Controls | ||||
C12 | 47 | 100 | 0 | 0 |
C13 | 22 | 100 | 0 | 5 |
C14 | 26 | 100 | 1 | 0 |
C15 | 81 | 100 | 9 | 11 |
C16 | 79 | 100 | 6 | 2 |
C17 | 80 | 100 | 0 | 8 |
Overall | 3 | 4 | ||
Obstructive azoospermia | ||||
O6 | 36 | 100 | 5 | 10 |
O7 | 29 | 100 | 2 | 22 |
Overall | 4 | 16 | ||
Non-obstructive azoospermia | ||||
HY1 | 40 | 88 | 6 | 26 |
HY2 | 24 | 100 | 8 | 7 |
HY8 | 34 | 100 | 10 | 16 |
MA1a | 34 | 22 | 63.6 | 72.7 |
Overall | 12 | 20 |
Patient . | Age (years) . | No. of pachytene cells analyzed . | Percentage of cells with incomplete synapsis . | Percentage of cells with at least 1 bivalent with 0 MLH1 . |
---|---|---|---|---|
Controls | ||||
C12 | 47 | 100 | 0 | 0 |
C13 | 22 | 100 | 0 | 5 |
C14 | 26 | 100 | 1 | 0 |
C15 | 81 | 100 | 9 | 11 |
C16 | 79 | 100 | 6 | 2 |
C17 | 80 | 100 | 0 | 8 |
Overall | 3 | 4 | ||
Obstructive azoospermia | ||||
O6 | 36 | 100 | 5 | 10 |
O7 | 29 | 100 | 2 | 22 |
Overall | 4 | 16 | ||
Non-obstructive azoospermia | ||||
HY1 | 40 | 88 | 6 | 26 |
HY2 | 24 | 100 | 8 | 7 |
HY8 | 34 | 100 | 10 | 16 |
MA1a | 34 | 22 | 63.6 | 72.7 |
Overall | 12 | 20 |
aSignificantly higher percentage of incomplete synapsis and significantly higher percentage of cells with at least one bivalent without MLH1 foci when compared with both groups.
Abbreviations: C, control; O, obstructive azoospermia; HY, hypospermatogenesis; MA, maturation arrest. Italicized entries point out men who had a significantly higher percentage of cells with at least 1 bivalent without MLH1 foci compared with the control group.
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