[Sex chromosomes and meiosis]. / Chromosomes sexuels et méiose.

Sex chromosome behaviour fundamentally differs between male and female meiosis. In oocyte, X chromosomes synapse giving a XX bivalent which is not recognizable in their morphology and behaviour from autosomal bivalents. In human male, X and Y chromosomes differ from one another in their morphology and their genetic content, leading to a limited pairing and preventing genetic recombination, excepted in homologous region PAR1. During pachytene stage of the first meiotic prophase, X and Y chromosomes undergo a progressive condensation and form a transcriptionally silenced peripheral XY body. The condensation of the XY bivalent during pachytene stage led us to describe four pachytene substages and to localize the pachytene checkpoint between substages 2 and 3. We also defined the pachytene index (PI=P1+P2/P1+P2+P3+P4) which is always less than 0.50 in normal meiosis. XY body undergoes decondensation at diplotene stage, but transcriptional inactivation of the two sex chromosomes or Meiotic Sex Chromosome Inactivation (MSCI) persists through to the end of spermatogenesis. Sex chromosome inactivation involves several proteins, some of them were now identified. Two isoforms of the HP1 protein, HP1beta and HP1gamma, are involved in the facultative heterochromatinization of the XY body, but the initiation of this process involves the phosphorylation of the protein H2AX by the kinase ATR whose recruitment depends on BRCA1. Extensive researches on the inactivation of the sex chromosomes during male meiosis will allow to a better understanding of some male infertilities.

[Macrocephalic spermatozoa. What would be the impact on reproduction?]. / Les spermatozoïdes macrocéphales. Quels risques pour la fonction de reproduction ?

OBJECTIVE: We want to highlight the risk of infertility and failure of Assisted Reproductive Technologies due to the presence of macrocephalic spermatozoa (MS) in the sperm at rate equalling or superior to 20% in at least one semen analysis. PATIENTS AND METHODS: We did a retrospective analysis of 19 infertile patients presenting MS at average rate between 14.3 and 49.7%. For each patient, at least one semen analysis showed a MS rate equal or superior to 20%. We did an automated analysis of the spermatozoa surface for 13 patients and a detailed analysis of the MS morphology in 18 patients. Thirteen couples benefited of one or more IVF with or without ICSI. RESULTS: The semen analysis shows an impairment of one or more parameter of the sperm in all patients. Three morphological aspects for MS were highlighted: MS with irregular head, MS with regular head, and MS with multiple heads, with a dominance of irregular heads. The spermatozoa surface analysis shows a significant increase of the average surface and of the standard deviation (p<0.0001). The average rate of pregnancies by transfer is decreased compared to usual rates in our laboratories (13% versus 28%). DISCUSSION AND CONCLUSION: We want to sensitize biologist and clinical doctors to the existence of partial forms of this syndrome, which could be related to infertility with impaired sperm parameters and low pregnancy rates after FIV or ICSI.

[Genetic aspects of the teratozoospermia]. / Aspects génétiques de la tératozoospermie.

Recent mutation identification in well-known sperm defects gives proof that there are genetic causes of infertility. Familial forms and some features of the spermograms lead toward the genetic origin of these syndromes. For each syndrome, several clinical aspects and partial forms were described. In these latter, apparently normal spermatozoa coexist with those showing the phenotype of interest. Transmission electron microscopy is the better tool to characterize the specific details of each syndrome. The frequency of genetic teratozoospermia is weak, the most studied syndromes are the globozoospermia, the macrocephaly, the syndrome of decapitated spermatozoa and the dyplasia of the fibrous sheat. A mutation was identified for two from these syndromes, but the two mutations does not account for all the cases from each syndrome. The various clinical aspects observed for each syndrome suggest that either other mutations or other genes are probably involved in these spermatogenic failures. The use of spermatozoa from patients for intra cytoplasmic sperm injection (ICSI) may pose two problems: fertilization problems and genetic risk for the progeny, including chromosomic and genic risk. Except for total macrocephaly which is excluded from ICSI because of sperm chromosomal abnormalities, these syndromes are consistent with assisted fertilization, but with uncertain rates of fertilization and pregnancy.

Meiotic abnormalities in patients bearing complete AZFc deletion of Y chromosome.

BACKGROUND: We studied meiosis in three infertile patients presenting complete AZFc microdeletion and three controls. METHODS: Primary spermatocytes were immunolabeled with SCP3, BRCA1 and gammaH2AX. We quantified the leptotene, zygotene and pachytene stages, and pachytene abnormalities: asynapsis and fragmented and dotted synaptonemal complexes (SCs). RESULTS: SCP3 level was significantly higher in leptotene and zygotene (bouquet) stages in patients, suggesting AZFc may have a direct effect on early prophase. SCs were abnormal in 77.3% of pachytene nuclei of patients versus 30.8% of controls. The two groups differed significantly (P < 0.001) in asynapsed nuclei, fragmented SC and dotted SCs. In patients, asynapsis were short and limited to a few bivalents. Staging of pachytene nuclei based on the morphology of the XY pair with BRCA1 revealed a prevalence of early pachytene substages (70.7%) in patients. H2AX was normally phosphorylated. CONCLUSIONS: In the absence of the AZFc region, the transient zygotene stage is extended, and chromosome condensation is reduced. The low level of limited asynapsis, the normal H2AX staining and the incomplete loss of germ cells at the pachytene checkpoint indicate that the AZFc region is not critical for meiotic recombination. We suggest that the pachytene phenotype develops secondarily to a primary defect that influences meiosis.

Evolution of DNA strand-breaks in cultured spermatocytes: the Comet Assay reveals differences in normal and gamma-irradiated germ cells.

In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

Meiotic anomalies in infertile men with severe spermatogenic defects.

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.

HP1beta and HP1gamma, but not HP1alpha, decorate the entire XY body during human male meiosis.

During meiosis in male mammals, the X and Y chromosomes become heterochromatic and transcriptionally silent, and form the XY body. Although the HP1 proteins are known to be involved in the packaging of chromosomal DNA into repressive heterochromatin domains, their involvement in facultative heterochromatinization has not been precisely determined. Here, we analyse, for the first time in humans, the subcellular distribution of the heterochromatin protein HP1alpha, HP1beta and HP1gamma isoforms, in male pachytene spermatocytes, and the XY body facultative heterochromatin in particular. Our results demonstrate that HP1beta and HP1gamma, but not the HP1alpha isoforms, decorate the entire XY body in half the pachytene nuclei observed. In some nuclei, the XY body appears to be only partially labelled. In these cases, the HP1beta and HP1gamma signals are adjacent to the Yq12 constitutive heterochromatin and signal appears to originate in this region before spreading over the entire XY body. This distribution suggests that HP1beta and HP1gamma proteins, which are components of the constitutive heterochromatin, may also be involved in the facultative heterochromatinization of the XY body. Nevertheless, their absence from the early pachytene substage, even though the XY body is already condensed, suggests that these proteins are not involved in the initiation of this process.

Pregnancies after ICSI using sperm with abnormal head-tail junction from two brothers: case report.

We report ICSI pregnancies in two couples with a history of long standing primary infertility in which the sperm of the male partner were either acephalic or had abnormal head-midpiece attachments. The two couples, in which the men are brothers, underwent ICSI. Sperm were analysed by transmission electron microscopy and immunocytochemistry with an anti-MPM2 monoclonal antibody. The first couple underwent two ICSI cycles, each consisting of the injection of two mature oocytes and the transfer of two embryos. A successful pregnancy occurred after the second transfer and led to the birth to a healthy girl. The second couple underwent three ICSI cycles, each consisting of the injection of 18 oocytes and the transfer of two embryos; the last of these led to a triple ongoing pregnancy which included two identical twins. Caesarean section led to the birth of three fetal-growth restricted children. This case report demonstrates that ongoing pregnancies can be achieved in cases of abnormal development of the head-neck attachment. The genetic origin of this syndrome is generally accepted, but the phenotypic heterogeneity observed by light and electron microscopy among published cases suggests that there are a variety of genetic causes of this syndrome.

Organization of the X and Y chromosomes in human, chimpanzee and mouse pachytene nuclei using molecular cytogenetics and three-dimensional confocal analyses.

We used multicolour fluorescence in-situ hybridization on air-dried pachytene nuclei to analyse the structural and functional domains of the sex vesicle (SV) in human, chimpanzee and mouse. The same technology associated with 3-dimensional analysis was then performed on human and mouse pachytene nuclei from cytospin preparations and tissue cryosections. The human and the chimpanzee SVs were very similar, with a consistently small size and a high degree of condensation. The mouse SV was most often seen to be large and poorly condensed, although it did undergo progressive condensation during pachynema. These results suggest that the condensation of the sex chromosomes is not a prerequisite for the formation of the mouse SV, and that a different specific mechanism could be responsible for its formation. We also found that the X and Y chromosomes are organized into two separate and non-entangled chromatin domains in the SV of the three species. In each species, telomeres of the X and Y chromosomes remain clustered in a small area of the SV, even those without a pseudoautosomal region. The possible mechanisms involved in the organization of the sex chromosomes and in SV formation are discussed.

A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection.

OBJECTIVE: To develop a reliable and simple method allowing meiotic studies to be performed on testicular samples used for ICSI. DESIGN: Evaluation of meiotic abnormalities in patients with severe spermatogenic impairment. SETTING: Centre de Médecine de la Reproduction, Marseille. PATIENT(S): Two azoospermic men undergoing testicular biopsy for ICSI and one control individual with normal testicular histology. INTERVENTION(S): The immature germ cells from the patients came from testicular biopsy used for ICSI, after dispersal into a thin cell suspension. Cells were cytocentrifuged to obtain well-spread spermatocytes and then immunocytochemical techniques were performed. We used rabbit polyclonal antibodies against the specific meiotic proteins Cor1 and Syn1 and a human CREST anti-kinetochore antibody. MAIN OUTCOME MEASURE(S): Synapsis abnormalities in patients with severe spermatogenesis impairment. RESULT(S): Pachytene spermatocytes are easily analyzed with this technique, without damage of the axial core and synaptonemal complex. The loss of germ cells is limited. CONCLUSION(S): The cytocentrifugation method is the most suitable technique for meiotic studies in patients with severe spermatogenic failure, because it can be used on the testicular cell suspension remaining after ICSI with testicular spermatozoa.

[Morphological aspect of embryos obtained after fertilization in vitro for male factor or ICSI]. / Aspect morphologique des embryons obtenus après une fécondation in vitro pour indication masculine ou ICSI.

OBJECTIVE: In order to study the eventual impact on fertilization and embryo characteristics of the microinjection procedure we compared the quality of the embryos obtained by ICSI with those of in vitro fertilization with male factors (MF IVF). MATERIAL AND METHODS: One hundred thirty-four cycles of IVF treatment (group 1) were selected with oligoasthenozoospermia according to WHO criteria with a total number of motile spermatozoa between 500,000 and 1 million. One thousand eighty-eight mature oocytes and 486 embryos were obtained. One hundred forty-three cycles of intracytoplasmic sperm injection (group 2) were performed in couples whose in vitro fertilization was imparticable because of extreme sperm impairment. One thousand one hundred forty-seven mature oocytes were injected and 626 embryos were obtained. RESULTS: In group 1, the pregnancy rate per embryo transfer and the implantation rate were respectively 22.7% and 12.3%. In group 2, the pregnancy rate per embryo transfer was 37.1% and the implantation rate was 17%. The statistical analysis of the embryos obtained in the two different groups did not demonstrate any difference in the distribution of the more regular and less fragmented embryos (group A) and those of the more irregular and fragmented embryos (group B). No statistical difference was demonstrated in the chronology of the division of these embryos (groups 1 and 2). CONCLUSION: The pregnancy rate by cycle and by transfer reported by ICSI (p < 0.003 and p < 0.015 respectively) could be related to a significantly higher mean number of transferred embryos (2.65 vs 2.02) in probable relation with a higher cleavage rate (p < 0.00001).

Bivalent 15 regularly associates with the sex vesicle in normal male meiosis.

Using fluorescent in-situ hybridization, we investigated the positioning of different human bivalents at the pachytene stage of normal male meiosis. We showed that, in about 35% of nuclei, the pericentromeric region of bivalent 15 is closely associated with the sex vesicle (SV). This behaviour may be linked to the presence of three domains in the pericentromeric region of chromosome 15: a large imprinted domain, a nucleolar organizing region (NOR), and a heterochromatic block. In order to define the domains of chromosome 15 involved in this association, we analysed the meiotic behaviour of other bivalents with similar domains: human bivalent 11 and mouse bivalent 7, bearing imprinted domains, other human acrocentric bivalents bearing a NOR, and the human bivalents 1, 9 and 16 containing a heterochromatic region. None of these bivalents were as frequently associated with the SV as the human bivalent 15. Nevertheless, we suggest that the bivalent 15 heterochromatin may be responsible for the association because of two properties: its telomeric location on chromosome 15 and its strong sequence homology with the Yq heterochromatin. This phenomenon could explain the high frequency of translocations between the chromosome 15 and the X or Y chromosomes.

Failure of pregnancy after intracytoplasmic sperm injection with decapitated spermatozoa: case report.

The case of a couple with a history of long standing primary infertility is reported in which the man presented with a decapitated sperm defect. The woman had a normal history and presented with normal clinical characteristics. The couple underwent one unsuccessful conventional in-vitro fertilization (IVF). Subsequently, embryos were obtained and transferred after assisted fertilization attempts: in all, three subzonal inseminations and four intracytoplasmic sperm injections. A total of 49 mature oocytes was injected in both studies, 25 embryos obtained and 20 embryos transferred, three of them after freezing and thawing. Despite the good embryo morphology, implantation was unsuccessful and no pregnancy occurred. The failure of implantation may have resulted from an arrest in early embryonic development related to the sperm anomaly. One hypothesis is that transferred embryos may carry a chromosomal imbalance that prevents them from progressing to the blastocyst stage. Nevertheless, we cannot exclude the possibility that the woman is responsible for the implantation failure. Co-culture associated with a further attempt could provide information regarding the ability of embryos to progress to the blastocyst stage and implant.

Rapid chromosomal analysis of germ-line cells by FISH: an investigation of an infertile male with large-headed spermatozoa.

A rapid fluorescence in-situ hybridization (FISH) technique was used for direct chromosomal analysis on germ cells from an infertile male with large-headed spermatozoa. The interphase chromosomes were fluorescently-labelled using an extremely bright cyanine dye during a 5-15 min FISH procedure. Germ cells were analysed using a battery of chromosome-specific DNA probes in several consecutive rapid FISH experiments. It was found that the majority of large-headed spermatozoa contained a diploid chromosome number probably due to errors in meiosis I or II divisions, whereas the majority of spermatozoa with normal sized heads are haploid and may be utilized for selective in-vitro fertilization procedures. Rapid FISH may be useful for the detection of major chromosomal aneuploidies in germ cells as an alternative technique to standard or multicolour FISH, and may find an additional application for the chromosomal analysis of human preimplantation embryos.

[Pachytene stage of meiosis in an infertile man carrying a reciprocal translocation between two acrocentric chromosomes]. / Etude du stade pachytène de la méiose chez un homme infertile porteur d'une translocation réciproque entre deux chromosomes acrocentriques.

Translocations involving acrocentric chromosomes are frequent in chromosomal male infertility. Robertsonian translocations are usually concerned whereas reciprocal translocations between acrocentric chromosomes are rarely encountered. The first reciprocal translocation involving the long arms of the two acrocentric chromosomes 13 and 14 [46,XY,t(13,14)(q33,q22)] is presented in this paper. The patient presents a severe oligoasthenospermia; testicular histology shows an important impairment of spermatogenesis. The quadrivalent is present in all the pachytene nuclei analyzed. A straight contact between the sex vesicle and the quadrivalent was found in 40.9% of the nuclei. A frequent asynapsis is localized at the breakpoint region (33.8%). The XY-autosome association was obtained by the central asynapsis and/or by the terminal chromomeres of the acrocentric chromosomes involved in the translocation.

[Quality of embryos in unexplained sterility]. / Qualité des embryons dans les stérilités inexpliquées.

In our study, the rate of pregnancy by transfer and puncture was not significantly different in unexplained and in tubal infertility, but the mean number of transferred embryos was significantly higher in the first group. To explain these data, we compared the quality of embryos in 32 punctures realized among 29 women with unexplained infertility and in 171 punctures planned among 156 women with tubal infertility. The percentage of embryos with 4 or more blastomeres was significantly lower in the unexplained infertility group than in the pure tubal infertility group.

[Genetic aspects of male infertility]. / Aspects génétiques de la stérilité masculine.

Cytogenetic investigations in sterile males showed that autosomal and sex chromosome anomalies can be responsible of the defect of spermatogenesis. Specific alteration of genes controlling spermatogenesis is excluded because all chromosomes are involved in the anomaly and breakpoints are distributed at random. In the aim to explain the spermatogenic failure, three mechanisms are proposed: X-autosome interaction, asynapsis, somatic lesion of the gonad, each mechanism might act alone or with the other. Beside chromosomal infertility, meiotic or sperm anomalies suggest the presence of gene mutations which interfere with the evolution of spermatogenesis or lead to the formation of abnormal spermatozoa. Insertion of technics of molecular biology in our researches could demonstrate the existence of these mutations; the same technics will allow to resolve the problem of the failure of a germ cell bearing a chromosomal anomaly particularly when this anomaly is balanced and involves the autosomes and not the sex chromosomes.

[Male infertility of chromosomal origin]. / L'infertilité masculine d'origine chromosomique.

Cytogenetic studies in infertile men showed that chromosomal anomalies were more frequent in these patients than in general population. Sex chromosome anomalies, specially 47, XYY karyotype, are predominant by their frequency at the severity of testicular impair. Nevertheless, balanced autosomal rearrangements can also induce spermatogenic failure; they were of great interest in perfecting the hypothesis which try to explain germ cells atresia. Three mechanisms are proposed: --X-autosome interaction; --synaptic failure; both could lead to a metabolic disorder and death of the germ cell; --somatic lesions of the gonad could also induce this degenerative process. Techniques of molecular biology joined up to cytogenetic investigations of meiosis will lead to a better understanding of the chromosomal male sterility.

Infertility in human males with autosomal translocations. II. Meiotic studies in three reciprocal rearrangements, one showing tertiary monosomy in a 45-chromosome individual and his father.

The meiotic prophase behavior of three human reciprocal autosomal translocations is presented. Each translocation was ascertained among men attending an infertility clinic. Two involved chromosomes 3 and 5, with breakpoints in different places. Quadrivalents were seen in every cell. The third translocation was a rare t(11q;15q) rearrangement in a 45-chromosome individual with tertiary monosomy. The long product of the translocation was retained in the karyotype over two generations of the family, the short product having been lost. At meiotic prophase, a trivalent was seen in every cell; in 60% of the nuclei, the short arm of the trivalent was closely associated with the XY bivalent. The transmission and phenotypic effects of tertiary monosomy in man and the mouse are discussed.