Meiotic anomalies in infertile men with severe spermatogenic defects

M.R. Guichaoua1,2,4, J. Perrin1,2, C. Metzler-Guillemain2, J. Saias-Magnan2, R. Giorgi3 and J.M. Grillo1,2

1 Laboratoire de Biogénotoxicologie et Mutagenèse Environnementale (EA1784), IFR PMSE112, Faculté de Médecine Timone, 27, Boulevard Jean Moulin, 13385, Marseille cedex 05, 2 Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5 and 3 Laboratoire d'enseignement et de recherche sur le traitement de l'Information Médicale (LERTIM), Faculté de Médecine, 27, Boulevard Jean Moulin, 13385 Marseille cedex 05, France

4 To whom correspondence should be addressed: Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5, France. Email: mguichaoua{at}ap-hm.fr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: This study was aimed at evaluating the rate of pairing failure in pachytene spermatocytes of patients presenting either an obstructive (O) or a non-obstructive (NO) infertility. METHODS: Forty-one patients and 13 controls underwent testicular biopsy. Among the patients, 19 had an O infertility and 22 a NO infertility. Preparations of all patients and controls were Giemsa-stained, and synaptonemal complexes from nine of these patients and one control were immunostained. RESULTS: In all, 2931 pachytene nuclei were analysed. The mean rate of asynapsed nuclei from the NO group (25.4%) was significantly higher than that of the O group (9.8%). There was no significant difference between the O group and the controls (10.6%). Immunocytochemistry showed that the number of pachytene nuclei decreased from the early to late pachytene sub-stage in all patients. Two NO patients, one azoospermic and one oligozoospermic, had a high percentage of asynapsed nuclei (86 and 91.8% respectively); one of these patients also presented a precocious localized separation of sister chromatids. CONCLUSION: high levels of extended asynapsis could arise from a primary meiotic defect which may be responsible for 9% of the NO male infertilities at our centre. The prevalence of early pachytene substages suggests that the pachytene checkpoint is localized at the mid-pachytene stage in humans.

Key words: asynapsis/meiosis/pachytene checkpoint/pachytene stage/spermatogenic failure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cytogenetic analysis of meiotic chromosomes in infertile men reveals a range of abnormal chromosome behaviour at each stage of meiosis (Egozcue et al., 2000Go). Lange et al. (1997)Go and Vendrell et al. (1999)Go found meiotic abnormalities in respectively 50 and 57.8% of men with highly abnormal sperm analysis, this incidence being ~5–6-fold higher than for the general infertile population. They also observed that the severity of the spermatogenic impairment was directly related to the incidence of meiotic chromosome abnormalities. However, the causes of the meiotic abnormalities and their relationship to spermatogenic failure are not clear. In most cases, meiotic arrest is thought to be secondary to a defective testicular environment that could be the consequence of a variety of aetiologies. Nevertheless, early meiotic studies carried out in men suggested the existence of meiotic mutations in humans (Hulten et al., 1970Go, 1974Go; Pearson et al., 1970Go; Templado et al., 1976Go, 1978Go, 1981Go; Vidal et al., 1982Go), and it has been proposed that meiotic chromosome abnormalities could explain the infertility in 6–8% of patients (Egozcue et al., 1983Go; De Braekeleer and Dao, 1991Go). In humans, however, few genes have been identified that express a meiosis-specific protein during the meiotic prophase of spermatogenesis. Even less is known about human meiotic mutations which lead to male infertility. Other factors, such as genotoxis, may induce infertility by causing impaired meiosis. Here we report the results of a retrospective meiotic study of 41 infertile men presenting either azoospermia or severe oligozoospermia, and 14 controls. We focused on the pairing and recombination behaviour of the homologous chromosomes at pachytene. For each patient and control, we determined the percentage of nuclei presenting asynapsed bivalents and we examined the possibility that the meiotic anomalies detected in some of our patients might be the primary cause of the infertility.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and controls
Testicular material was obtained from 41 patients aged from 24 to 46 years. Patients underwent testicular biopsy either as part of their infertility evaluation (32 patients) or for ICSI (nine patients). Characteristics of the patients are summarized in Tables I and II. Nineteen patients presented an obstructive infertility (O group) and 22 presented a non-obstructive infertility (NO group). They presented either an azoospermia or a severe oligozoospermia <2 x 106/ml. In the O group, patients were selected only if FSH level, karyotype and testicular volume were normal. Two of these patients presented a secondary infertility (patients TeH 34 and TeH 88), the other patients presented a primary infertility. In the NO group the FSH level was normal in 13 patients (≤10 IU/l), >10 IU/l in nine patients (from 11.6 to 45 IU/l). All patients from this group presented a primary infertility. One patient of the NO group had an abnormal karyotype: a mosaic 46,XY/47,XXY (TeH 8). Patient TeH 78 showed a pericentric inversion of chromosome 9. Controls were 13 fertile men aged 50–75 years who underwent orchidectomy as part of their treatment for prostatic carcinoma from 1980 to 1986. They were selected on the bases of abundant sperm and good quality meiotic cells in the testis.


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Table I. Characteristics of the patients from the obstructive group

 

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Table II. Characteristics of the patients from the non-obstructive group

 
Spreading and staining of the germ cells
Testicular fragments from the patients and controls were treated using the air-drying technique previously described by Luciani et al. (1975)Go to obtain well-spread germ cells at the first meiotic prophase; preparations were Giemsa-stained.

Immunofluorescence
Meiotic cells from nine of the above patients (five in the NO group and four in the O group) and one control were immunostained according to the technique described by Metzler-Guillemain and Guichaoua (2000)Go. Two antibodies were applied: (i) a rabbit anti-hamster synaptonemal complex polyclonal antibody which recognizes the Cor1 protein of the axial core/lateral element of the synaptonemal complex (SC) at a dilution of 1/1000 (kindly provided by Peter Moens and Barbara Spyropoulos); (ii) an anti-kinetochore serum from a patient having a CREST syndrome was used at dilution of 1/20 (kindly provided by Marielle Sanmarco). Two secondary antibodies (Sigma) were applied: a fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG at a dilution of 1/200 in phosphate-buffered saline (PBS) and a tetramethylrhodamine B isothiocyanate (TRITC)-conjugated goat anti-human IgG at a dilution of 1/16 in PBS for 1 h at 37°C.

Microscope analysis
After Giemsa staining, pachytene nuclei were analysed using a Zeiss Axioplan 2 photomicroscope (Zeiss, Germany) with a x 100 Plan apo objective. Analysis of immunofluorescent preparations was performed using the same microscope equipped with an epifluorescent system. Selected nuclei were then examined with a confocal scanner Leica TCS 4D mounted on a Leica DMIRBE microscope (Leica, Germany).

The number of pachytene spermatocytes analysed per patient varied from 20 to 100, with the exception of patient TeH 21 (17 nuclei analysed) (Table II) depending on the richness of the individual preparations. The pachytene nuclei were selected and analysed on the basis of good spreading of the bivalents and sex chromosome configuration, allowing unambiguous evaluation of pairing failure. For each patient, we quantified the number of pachytene nuclei showing ‘asynapsis’, which is defined as the pairing defect of either homologous chromosomes or segments of homologous chromosomes. These nuclei were termed ‘asynapsed nuclei’. Immunostained pachytene nuclei were classified as early or late substages, according to the different morphologies of the XY pair, based on the classification of Solari (1980)Go.

Statistical analysis
For each patient, we evaluated an ‘index of asynapsis’ which corresponded to the ratio: number of nuclei with asynapsed bivalents/number of nuclei analysed. We then used the Mann and Whitney U-test. We compared the results of the meiotic studies from the obstructive (O) group with the non obstructive (NO) group, and with controls. Statistical analysis was performed using SPSS 11.1 software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Testicular histology
In all men in the O group (Table I) the testicular histology showed the presence of germ cells at all stages of spermatogenesis in the majority of the tubules and abundant sperm production in the testis. Nervertheless, in one patient (TeH24) which was azoospermic due to a bilateral absence of vas deferens, hyperchromatosis of the germ cell nuclei was related to the obstacle of genital ducts. Interstitial tissue was normal in all patients.

Severe alterations of spermatogenesis were observed in all patients of the NO group (Table II). All men produced very few or no sperm in the testis. Four patients (TeH 9, TeH 17, TeH 21 and TeH 46) had Sertoli cells only at the histological level. In all the other patients, numerous Sertoli cell only tubules were seen on the preparations; the other tubules showed hypospermatogenesis with few sperm. Four patients (TeH 2, TeH 16, TeH 52, TeH 66) were mosaic at the histological level, also showing tubules with normal spermatogenesis. Two patients (TeH 3 and TeH 10) had a maturation arrest, showing interrupted spermatogenesis at round spermatid stage. No sperm were seen in these two patients at the histological level, but both had sperm in the ejaculate. Impaired fibrous and/or oedematous interstitial tissue, and hyalinose of the tubule walls were reported in the majority of the patients.

Meiotic study
The results are summarized in Table III. A total of 2931 pachytene nuclei were analysed, 1354 for the O group and 1577 for the NO group. The mean number of pachytene nuclei analysed per patient was 58 for both groups. The mean rates of asynapsed nuclei in the O group and the NO group were 9.8 and 25.4% respectively, and the difference between the two groups was statistically significant (P<0.001). In total, 855 nuclei were analysed for the 14 controls, with a mean of 59 nuclei analysed per control. The mean rate of asynapsed nuclei in this group was 10.6%.


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Table III. Numbers and rates of asynapsed pachytene nuclei observed after Giemsa staining and immunofluorescence, from obstructive (O) and non-obstructive (NO) patients and controls

 
Giemsa staining
The meiotic cells of all patients and controls were analysed with this technique. All patients who were Sertoli cell only at the histological level (TeH 9, TeH 17, TeH 21 and TeH 46) nevertheless showed primary spermatocytes at the cytological level. The number of pachytene nuclei analysed was the lowest in patient TeH 21. Completely asynapsed nuclei were rare and were observed in a very small number of patients. Rare short terminal asynapsis, which may be an artefact of the spreading technique, was not taken into account in this study. In all but two patients (TeH 3 and TeH 10), asynapsis was limited to a fairly short region of one or a few bivalents. In the control group, the percentage of asynapsed nuclei varied from 8.6 to 14.2%, with a mean of 10.8% (Table III). In the O group, the percentage of asynapsed nuclei varied from 0% to 23.5%, with a mean of 10.7% (Tables I and III). There was no significant difference between the O group and the controls (10.7 versus 10.8%, P=0.76). In the NO group, the percentage of asynapsed nuclei varied from 4 to 91.8%, with a mean of 27.4% (Tables II and III). The difference between the percentages of asynapsis in the O group and the NO group was statistically significant (P<0.001). Two exceptional patients (TeH 3 and TeH 10) in the NO group presented a particularly high percentage of asynapsed nuclei (86 and 91.8% respectively). Both patients showed extensive asynapsis on several or all bivalents; in the case of patient TeH 3, there was precocious localized separation of sister chromatids (PSSC), on several bivalents, in 11% of the pachytene nuclei (Figure 1A). In addition to these extended asynapses, the sex chromosomes in both patients were uncondensed (lack of visible XY body) in 87 and 65.3% of nuclei respectively, and all autosomal bivalents were uncondensed in 91 and 79.5% of nuclei respectively (Figure 1B). No normal pachytene nuclei were found in these two patients but both had sperm (1 x 106 to 104 sperm/ml for the first patient and 104 sperm/ml to azoospermia for the second patient). Both patients had spermatid block on histological analysis. Thus in this study, the frequency of patients with evidence of a specific meiotic defect was 4.8% of the patients analysed and 9% of the NO infertile men.



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Figure 1. (A, B) Abnormal pachytene nuclei obtained from patient TeH3 after Giemsa staining. (A) Pachytene nucleus showing extended asynapsis (long arrows) and separation of sister chromatids (short arrows). Autosomal bivalents and the XY pair were uncondensed. (B) Pachytene nucleus showing fully synapsed uncondensed bivalents. Scale bar = 5 µm. (C, D) Epifluorescent micrographs of pachytene nuclei. Synaptonemal complex (SC) and axial elements are immunostained with a polyclonal antibody recognizing Cor1 proteins (green). (C) Type III pachytene nucleus (type III XY pair, long arrow). Limited asynapsis is visible on an autosomal bivalent (short arrow). (D) Extended asynapsed nucleus, XY pair could not be identified. (E, F) Normal pachytene nuclei analysed by confocal scanner. SC and axial elements are immunostained with a polyclonal antibody recognizing Cor1 proteins (green). Kinetochores are immunostained by an anti-kinetochore serum from a patient with CREST syndrome (red). Both configurations of the X and Y chromosomes shown (arrows) allow early and late pachytene nuclei to be distinguished. (E) Late pachytene spermatocyte I, showing a dense XY body (type IV). (F) Early pachytene spermatocyte I, showing well-individualized X and Y axial elements (type I). Scale bar = 5 µm.

 
Excluding these two exceptional patients from the NO group, the percentage of asynapsed nuclei varied from 4 to 48%, with a mean of 21.2%; the difference between the O group and the NO group remained statistically significant (10.7 versus 21.2%) (P<0.001).

Immunofluorescence
Nine patients and a control were analysed using this staining, and the characteristics of the asynapsis were as observed with Giemsa staining (Figure 1C and D). The mean number of pachytene nuclei analysed per patient was 70 for the O group and 63 for the NO group. In the O group, the percentage of asynapsed nuclei varied from 2.4 to 9.5%, with a mean of 6%; in the NO group, the percentage of asynapsed nuclei varied from 7.6 to 26.9%, with a mean of 17.2% (Tables III and IV). The difference between the two groups was statistically significant (P=0.032). The percentage of asynapsis in the control for this protocol was 11.1%.


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Table IV. Numbers and rates of asynapsed pachytene nuclei identified by immunofluorescence in four patients from the obstructive group and five patients from the non-obstructive group

 
The immunostained sex bivalents were observed to have one of the four configurations (types I, II, III and IV) (Figure 1C, E and F), corresponding to four of the six configurations described by Solari (1980)Go using the microspreading technique. Types I and II XY pairs occur during early to mid-pachytene, while types III and IV occur during later pachytene substages. In all patients analysed, the number of early pachytene nuclei (stages I and II) observed in the preparations was higher than the number of late pachytene nuclei (stages III and IV), with means of 76.6 versus 23.1% respectively for the O group, and 77.7 versus 22.2% respectively for the NO group (Table V). Conversely, in the control, early pachytene nuclei were less frequent than late pachytene nuclei (46.0 versus 53.9%).


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Table V. Staging of pachytene cells, based on the morphology of the XY pair by immunofluorescence

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this retrospective study, we show that pairing failure is a general phenomenon during meiosis, and in a proportion of cases (4.8% in this study) high levels of extended asynapsis which could arise from a primary meiotic defect may be responsible for the infertility. Indeed, almost all the infertile patients, and all controls, present a moderate frequency of asynapsed nuclei. Only one O patient showed 0% of asynapsed nuclei (TeH 49). As we expected, the mean rate of asynapsis for the NO group was significantly higher than that for the O group (Table III). Nevertheless, one of the most interesting findings in our study was that two patients in the NO group (TeH 3 and TeH 10) showed an exceptionally high rate of homogeneous meiotic errors (>85%) and a spermatid arrest. These high levels of extended asynapsis could only arise from specific defects affecting one of the two crucial steps of meiosis, recombination or pairing, possibly through the presence of mutations in key meiotic genes (Egozcue et al., 2000Go; Judis et al., 2004Go). In our study, precocious sister chromatid separation (PSSC) in 11% of nuclei in patient TeH 3, but 0% in TeH 10, suggests that there are distinct origins for the meiotic defects in these two patients, with a more severe phenotype resulting in patient TeH 3.

It was suggested by de Boer et al. (2004)Go that zygotene may often be misinterpreted as asynapsed pachytene, while de Vries et al. (1999)Go refer to the possibility of an ‘extended zygotene stage’. Sex vesicle analysis, however, does allow an exact identification of the pachytene stage, and applying either interpretation, accumulation of zygotene or early pachytene spermatocytes is an indication that progression of meiosis is defective. It is likely that in most cases this is a secondary effect, and this is clearly shown by our observation that early stages are more common than late in the members of the two groups tested by immunocytochemistry. Indeed, in this study, staging of pachytene cells, based on the morphology of the XY chromosome pair, showed a prevalence of early pachytene substages in the O group (83.5%) and in the NO group (78.1%), whereas in the control group, late pachytene nuclei were in the majority (53.9%), as has previously been reported by Solari (1980)Go and de Boer et al. (2004)Go. Thus, although the asynapsis rates were the same in both the O group and in controls, the finding that early pachytene substages consistently prevail in both the O group and the NO group suggests that meiotic germ cell development is impaired even in cases of obstructive infertility. Moreover, the prevalence of early pachytene nuclei in patients of the two groups suggests that the pachytene checkpoint is localized in mid-pachytene in humans and acts effectively in patients with meiotic anomalies. However, the fact that sperm were present at the time of meiotic study in patient TeH 3 suggests that the pachytene checkpoint is not an absolute barrier. Indeed, with the exception of Aran et al. (2003)Go, the majority of studies show that abnormal meiotic cells can complete spermatogenesis. A significant increase of aneuploidy, disomy and diploidy in sperm was shown by sperm FISH analysis for infertile men (Moosani et al., 1995Go; Bernardini et al., 1998Go; Pang et al., 1999Go; Calogero et al., 2001Go; Schmid et al., 2003Go) and for a men carrying a mutant allele of the mismatch repair gene, hMSH2 (Martin et al., 2000Go). In the same way, fertilization and pregnancy rates in obstructive azoospermia were higher than those achieved in non-obstructive azoospermia (Palermo et al., 1999Go; Balaban et al., 2001Go; Vernaeve et al., 2003Go). Thus, qualitative and quantitative analyses of meiotic abnormalities are essential for the identification of meiotic abnormalities which could be directly responsible for the spermatogenic failure and thus reveal new aetiologies of male infertility. The panel of antibodies which detects individual protein components at different stages of meiosis provides a valuable tool for the detection and interpretation of abnormal meiotic profiles (Sun et al., 2004Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Prof. P.Moens, Dr B.Spyropoulos, for the generous gift of the Cor1 antibody; Dr M.Sanmarco for the gift of anti-kinetochore serum; Prof. D Rossi and Prof. G Serment for the gift of testicular samples; Prof. J. Sampol and R. Pistoresi for the confocal analysis; Dr M.Mitchell for his assistance with the English; D.Daïoglou, C.Metton and M.Fraterno for their technical assistance. This work was supported by grants from the Assistance Publique of Marseille and the French Cancer Research Association (ARC).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 15, 2004; resubmitted on February 22, 2005; accepted on February 24, 2005.





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