Marie-Roberte Guichaoua

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility

Background: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. Methods: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. Results: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. Conclusions: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

Complete deletion of the AZFb interval from the Y chromosome in an oligozoospermic man

BACKGROUND Deletion of the entire AZFb interval from the Y chromosome is strictly associated with azoospermia arising from maturation arrest during meiosis. Here, we describe the exceptional case of an oligozoospermic man, 13-1217, with an AZFb + c (P5/distal-P1) deletion. Through the characterization of this patient, and two AZFb (P5/proximal-P1) patients with maturation arrest, we have explored three possible explanations for his exceptionally progressive spermatogenesis. METHODS AND RESULTS We have determined the precise breakpoints of the deletion in 13-1217, and shown that 13-1217 is deleted for more AZFb material than one of the AZFb-deleted men (13-5349). Immunocytochemical analysis of spermatocytes with an antibody against a synaptonemal complex component indicates synapsis to be largely unaffected in 13-1217, in contrast to 13-5349 where extended asynapsis is frequent. Using PCR-based analyses of RNA and DNA from the same testicular biopsy, we show that 13-1217 expresses post-meiotic germ cell markers in the absence of genomic DNA and transcripts from the AZFb and AZFc intervals. We have determined the Y chromosome haplogroup of 13-1217 to be HgL-M185. CONCLUSIONS Our results indicate that the post-meiotic spermatogenesis in 13-1217 is not a consequence of mosaicism or retention of a key AZFb gene. On the contrary, since the Hg-L Y chromosome carried by 13-1217 is uncommon in Western Europe, a Y-linked modifier locus remains a possible explanation for the oligozoospermia observed in patient 13-1217. Further cases must now be studied to understand how germ cells complete spermatogenesis in the absence of the AZFb interval.

Occupational Exposures Obtained by Questionnaire in Clinical Practice and Their Association With Semen Quality

In industrial countries, evidence suggests that semen quality has been steadily decreasing over the past 5 decades. We employed a short questionnaire to examine the association between self-reported physical or chemical occupational exposures and semen quality. The study included 402 men consulting for couple infertility (314 with oligospermia, asthenospermia, or teratospermia and 88 with normal semen; World Health Organization criteria). Exposure effects on global sperm quality and total sperm count, sperm motility, and sperm morphology were investigated. We found significant associations between semen impairment and occupational risk factors such as exposure to heavy metals (adjusted odds ratio [OR] = 5.4; 95% confidence interval [CI], 1.6-18.1), solvents (OR = 2.5; 95% CI, 1.4-4.4), fumes (OR = 1.9; 95% CI, 1.1-3.4), and polycyclic aromatic hydrocarbons (OR = 1.9; 95% CI, 1.1-3.5). Exposure to pesticides or cement was nearly significant (OR = 3.6; 95% CI, 0.8-15.8, and OR = 2.5; 95% CI, 0.95-6.5, respectively). Physical risk factors were associated with some sperm anomalies, such as mechanical vibrations with oligospermia and teratospermia as well as excess heat and extended sitting periods with impaired motility. Exposure to ionizing radiation and electromagnetic fields was not associated with semen impairment; these results, however, may be skewed, because very few subjects reported such exposure. Despite the small dataset, self-reported exposures were correlated with semen impairment. This approach may be recommended in routine clinical practice to seek relationships between occupational exposures to reprotoxic agents and impaired semen parameters. This knowledge would allow preventive measures in the workplace to be established and could be complemented by the use of biomarkers to better characterize exposure to chemical substances and their spermiotoxic effects.

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.

Exposure to Low-Dose Bisphenol A Impairs Meiosis in the Rat Seminiferous Tubule Culture Model: A Physiotoxicogenomic Approach

Background Bisphenol A (BPA) is one of the most widespread chemicals in the world and is suspected of being responsible for male reproductive impairments. Nevertheless, its molecular mode of action on spermatogenesis is unclear. This work combines physiology and toxicogenomics to identify mechanisms by which BPA affects the timing of meiosis and induces germ-cell abnormalities. Methods We used a rat seminiferous tubule culture model mimicking the in vivo adult rat situation. BPA (1 nM and 10 nM) was added to the culture medium. Transcriptomic and meiotic studies were performed on the same cultures at the same exposure times (days 8, 14, and 21). Transcriptomics was performed using pangenomic rat microarrays. Immunocytochemistry was conducted with an anti-SCP3 antibody. Results The gene expression analysis showed that the total number of differentially expressed transcripts was time but not dose dependent. We focused on 120 genes directly involved in the first meiotic prophase, sustaining immunocytochemistry. Sixty-two genes were directly involved in pairing and recombination, some of them with high fold changes. Immunocytochemistry indicated alteration of meiotic progression in the presence of BPA, with increased leptotene and decreased diplotene spermatocyte percentages and partial meiotic arrest at the pachytene checkpoint. Morphological abnormalities were observed at all stages of the meiotic prophase. The prevalent abnormalities were total asynapsis and apoptosis. Transcriptomic analysis sustained immunocytological observations. Conclusion We showed that low doses of BPA alter numerous genes expression, especially those involved in the reproductive system, and severely impair crucial events of the meiotic prophase leading to partial arrest of meiosis in rat seminiferous tubule cultures.

Homozygous deletion of SUN5 in three men with decapitated spermatozoa

A recent study of 17 men with decapitated spermatozoa found that 8 carried two rare SUN5 alleles, and concluded that loss of SUN5 function causes the acephalic spermatozoa syndrome. Consistent with this, the SUN5 protein localises to the head-tail junction in normal spermatozoa, and SUN proteins are known to form links between the cytoskeleton and the nucleus. However, six of the ten SUN5 variants reported were missense with an unknown effect on function, and only one man carried two high confidence loss-of-function (LOF) alleles: p.Ser284* homozygozity. One potential exonic splice mutation, homozygous variant p.Gly114Arg, was not tested experimentally. Thus, definitive proof that loss of SUN5 function causes the acephalic spermatozoa syndrome is still lacking. Based on these findings, we determined the sequence of the SUN5 gene in three related men of North African origin with decapitated spermatozoa. We found all three men to be homozygous for a deletion-insertion variant (GRCh38 - chr20:32995761_32990672delinsTGGT) that removes 5090 base pairs including exon 8 of SUN5, predicting the frameshift, p.(Leu143Serfs*30), and the inactivation of SUN5. We therefore present the second case where the acephalic spermatozoa syndrome is associated with two LOF alleles of SUN5. We also show that the p.Gly114Arg variant has a strong inhibitory effect on splicing in HeLa cells, evidence that homozygozity for p.Gly114Arg causes acephalic spermatozoa syndrome through loss of SUN5 function. Our results, together with those of the previous study, show that SUN5 is required for the formation of the sperm head-tail junction and male fertility.

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.

Syndrome des spermatozoïdes macrocéphales polyflagelles et Assistance Médicale à la Procréation

RésuméLe syndrome des spermatozoïdes macrocéphales est classiquement décrit comme une atteinte de l’ensemble de la population gamétique. L’étiopathogénie de ce syndrome est encore mal déterminée, mais les anomalies méiotiques y sont constantes d’où l’appellation de Déficience de Division Méiotique (Meiotic Division Deficiency, MDD). La ou les mutations en cause ne sont pas déterminées. Plusieurs variants phénotypiques existent.Nous avons mis en évidence un nouveau variant de ce syndrome, basé sur l’observation de populations gamétiques en mosaïque dans lesquelles coexistent des spermatozoïdes macrocéphales et des spermatozoïdes de taille normale. L’observation de syndromes MDD accompagnés d’une histoire familiale de morts périnatales laisse supposer qu’une atteinte du mécanisme de division des cellules somatiques pourrait accompagner celui des cellules germinales. L’Assistance Médicale à la Procréation (AMP), et plus particulièrement l’injection intra cytoplasmique de spermatozoïde permet d’obtenir des grossesses chez des couples dont le conjoint est proteur de ce syndrome.Nous envisageons d’étudier des couples dont le conjoint présente une population de spermatozoïdes macrocéphales afin de définir précisément le phénotype morphologique du syndrome des spermatozoïdes macrocéphales en mosaïque (et regrouper les observations en différentes catégories), d’apprécier le type de transmission de ce syndrome par l’établissement d’arbres généalogiques et d’évaluer grâce à la technique d’hybridationin situ en fluorescence le pourcentage de spermatozoïdes de taille normale aneuploïdes. Nous suivrons le parcours de ces couples en AMP et nous rechercherons une incidence accrue de paucifécondation, l’absence d’implantation embryonnaire, de fausses couches spontanées ou de toute anomalie pouvant survenir au cours de la grossesse.AbstractThe first case of macrocephalic spermatozoa with multiple tails was described in 1977 by Nistalet al. In its classical form (type I), the syndrome comprises a combination of oligoasthenoteratozoospermia, sperm heads of increased size with irregular shapes and multiple flagellae. Fluorescentin situ hybridization (FISH) on sperm chromosomes reveals disorders such as diploidy, triploidy, tetraploidy or aneuploidy. A genetic origin is strongly suggested by the existence of familial cases.Although the pathophysiology has not been fully elucidated, meiotic disorders are constantly found giving this syndrome the name of Meiotic Division Deficiency (MDD). Several cellular structures may be involved: cytoskeleton disorders (spindle or centrosome) could give rise to abnormal chromosome distribution during meiosis and could explain irregular head shapes. Numerous phenotypes have already been described.We observed that a population of men attending our infertility center showed an incomplete form of the syndrome, with only a clone of macrocephalic germinal cells. This observation is more common than the classical type. FISH on the spermatozoa of these patients show that normalsized spermatozoa can be aneuploid, with a higher incidence than controls.The description of a family history of MDD with multiple perinatal deaths reveals that a somatic cell disorder can be associated. In the common type, the low sperm count found in MDD is not compatible with natural fertilization. Intracytoplasmic sperm injections (ICSI) have been performed for these patients, giving rise to pregnancies although fertilization and pregnancy rates were low. Embryos with an incorrect set of chromosomes could result from the injection of aneuploid spermatozoa, or could be due to a mutation affecting both germinal and somatic cell lines, thus explaining abnormal chromosome distribution in the very first segmentation divisions of the embryo.We plan to study couples attending our infertility center in which men show an increased percentage of macrocephalic spermatozoa, in order to precisely define the morphological phenotype of the mosaic macrocephalic spermatozoa syndrome. We will also assess the type of genetic transmission involved and will use FISH to estimate the percentage of normal-sized spermatozoa showing abnormal chromosome numbers. Follow-up of the couple is necessary for evaluation of fertilization and pregnancy rates in this syndrome.