Search

International audience; Purpose: Infertility in adolescents and young adult (AYA) survivors of malignant disease remains a major long-term adverse effect, but semen collection for fertility preservation in fertility centers is not always feasible and makes AYAs uncomfortable. We evaluated the feasibility of collecting sperm samples on the ward versus in fertility centers. Methods: Consecutive hospitalized AYA-aged male patients in the Hematology AYA unit (Saint-Louis Hospital, France) between August 2010 and June 2016 with hematological disease and indication of semen collection (n = 95) were included in this retrospective study. Semen quality was analyzed according to World Health Organization guidelines and was compared according to semen collection place: on the ward (n = 46) or in fertility center (n = 49). Results: The median age was median age 19.1 years (range: 13.7-33.3; interquartile range: 17.1-22.8) and 85 patients successfully collected semen. Sperm collection failure was ∼11% and was comparable between the two modalities as were main sperm quality characteristics (semen volume, sperm concentration, total sperm count, progressive motility and vitality, sperm morphology, and multiple anomalies index). Oligospermia was significantly higher in the samples obtained in fertility center (47.7%) than on the ward (26.8%), p = 0.047. Average frozen straws were comparable, 12.2 ± 6.4 on the ward versus 11.9 ± 6.3 in fertility center. Conclusion: Semen collection on the ward is feasible and would be particularly interesting for AYA male patients without altering semen quality characteristics.

International audience; Abstract Aim: To provide practice guidelines about fertility preservation (FP) in oncology. 93Methods: We selected 400 articles after a PubMed review of the literature (1987e2019). Recommendations: Any child, adolescent and adult of reproductive age should be informed about the risk of treatment gonadotoxicity. In women, systematically proposed FP counselling between 15 and 38 years of age in case of treatment including bifunctional alkylating agents, above 6 g/m2 cyclophosphamide equivalent dose (CED), and for radiation doses on the ovaries 3 Gy. For postmenarchal patients, oocyte cryopreservation after ovarian stimulation is the firstline FP technique. Ovarian tissue cryopreservation should be discussed as a first-line approach in case of treatment with a high gonadotoxic risk, when chemotherapy has already started and in urgent cases. Ovarian transposition is to be discussed prior to pelvic radiotherapy involving a high risk of premature ovarian failure. For prepubertal girls, ovarian tissue cryopreservation should be proposed in the case of treatment with a high gonadotoxic risk. In pubertal males, sperm cryopreservation must be systematically offered to any male who isto undergo cancer treatment, regardless of toxicity. Testicular tissue cryopreservation must be proposed in males unable to cryopreserve sperm who are to undergo a treatment with intermediate or severe risk of gonadotoxicity. In prepubertal boys, testicular tissue preservation is: - recommended for chemotherapy with a CED 7500 mg/m2 or radiotherapy 3 Gy on both testicles. - proposed for chemotherapy with a CED 5.000 mg/m2 or radiotherapy 2 Gy. If several possible strategies, the ultimate choice is made by the patient.

In these two short reports, the authors approach the issue of whether hydroxyurea (HU) use in young males has major irreversible effects on sperm production. Joseph et al analyzed and compared sperm parameters in male patients with sickle cell disease (SCD) who were exposed or not exposed to HU before puberty. They report semen abnormalities in all patients but no differences between groups. Independently, Gille et al provide evidence for the lack of in vivo HU-related decreases in the spermatogonial pool in biopsy specimens from young males with SCD but evidence for a negative effect of SCD itself. Together, these reports suggest that the use of HU in young males does not adversely affect fertility.

The shaping of the human embryo begins with compaction, during which cells come into close contact and form a tighter structure1,2. Assisted reproductive technology (ART) studies suggest that human embryos fail compaction primarily because of defective adhesion3,4. Based on our current understanding of animal morphogenesis5,6, other morphogenetic engines, such as cell contractility, could be involved in shaping the human embryo. However, the molecular, cellular and physical mechanisms driving human embryo morphogenesis remain uncharacterized. Using micropipette aspiration on human embryos donated to research, we have mapped cell surface tensions during compaction. This reveals a 4-fold increase of tension at the cellmedium interface while cell-cell contacts keep a steady tension. Comparison between human and mouse reveals qualitatively similar but quantitively different mechanical strategies, with human embryos being mechanically least efficient. Inhibition of cell contractility and cell-cell adhesion in human embryos reveal that only contractility controls the surface tension responsible for compaction. Interestingly, if both cellular processes are required for compaction, they exhibit distinct mechanical signatures when faulty. Analyzing the mechanical signature of naturally failing embryos, we find evidence that non-compacting embryos or partially compacting embryos with excluded cells have defective contractility. Together, our study reveals that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.

Male infertility is responsible for approximately half of all cases of reproductive issues. Spermatogenesis originates in a small pool of spermatogonial stem cells (SSCs), which are of interest for therapy of infertility but remain not well defined in humans. Using multiparametric analysis of the side population (SP) phenotype and the α-6 integrin, THY1, and β-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype β-2 microglobulin (β-2M)

Ongoing progress in genomic technologies offers exciting tools that can help to resolve transcriptome and genome-wide DNA modifications at single-cell resolution. These methods can be used to characterize individual cells within complex tissue organizations and to highlight various molecular interactions. Here, we will discuss recent advances in the definition of spermatogonial stem cells (SSC) and their progenitors in humans using the single-cell transcriptome sequencing (scRNAseq) approach. Exploration of gene expression patterns allows one to investigate stem cell heterogeneity. It leads to tracing the spermatogenic developmental process and its underlying biology, which is highly influenced by the microenvironment. scRNAseq already represents a new diagnostic tool for the personalized investigation of male infertility. One may hope that a better understanding of SSC biology could facilitate the use of these cells in the context of fertility preservation of prepubertal children, as a key component of regenerative medicine.

Study question Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? Summary answer JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. What is known already Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated. Study design, size, duration We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation. Participants/materials, setting, methods Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank). Main results and the role of chance Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation. Large-scale data N/A. Limitations, reasons for caution In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions. Wider implications of the findings Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples. Study funding/competing interests This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogenetique (ICIG). There are no competing interests.

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

International audience; PURPOSE:The aims of this study were to investigate the possible benefits of extending the culture of poor-quality day-2 embryos (PQE) versus good-quality embryos (GQE) and to identify factors associated with pregnancy and live birth when transferring frozen-thawed blastocysts originating from GQE and PQE.METHODS:This is a retrospective cohort follow-up study performed between November 2012 and February 2015 at the IVF Laboratory Unit of Cochin University Hospital (Paris, France) including 3108 day-2 supernumerary embryos resulting from 1237 IVF/ICSI cycles.RESULTS:Total blastulation rate was 67.2% from GQE and 48.7% from PQE. Percentage of good-quality blastocysts was 60.7 and 47.9% respectively including 14.7 and 7.3% top-quality blastocysts. A total of 150 blastocysts originating from GQE and 729 from PQE were frozen, and then, 37 and 164 were thawed and transferred respectively resulting in 19 (51.4%) and 61 (37.9%) clinical pregnancies with 13 (35.1%) deliveries from GQE and 32 (19.9%) from PQE (p = 0.046) without any difference in neonatal outcomes. Quality of blastocysts that resulted in live birth was similar in the two groups. Women < 35 years old and day-5 blastocyst expansion were predictive of pregnancy and live birth.CONCLUSIONS:(i) PQE are able to reach the blastocyst stage, to implant, and to give healthy babies and (ii) women age and day of blastocyst expansion are predictive of pregnancy and live birth.

STUDY QUESTION The aim of this study was to evaluate the live birth rate (LBR) after frozen-thawed Day 5 (D5) and Day 6 (D6) blastocyst transfers. SUMMARY ANSWER LBR following frozen-thawed blastocyst transfer is significantly lower with D6 than with D5 blastocyst regardless of embryo quality. WHAT IS KNOWN ALREADY During fresh embryo transfer cycles, pregnancy rates (PR) are significantly higher when transferring blastocysts expanded on D5 compared with slow developing blastocysts (D6). In programmed thawed blastocyst transfer (TBT) cycles, the same clinical outcomes should be expected when transferring D5 or D6 blastocysts because of endometrial/embryonic synchronization due to hormonal priming of endometrial receptivity. However, the impact of delayed blastocyst expansion at D6 on clinical outcomes remains unclear. Some reports have shown higher PRs after D5 TBT compared with those of D6, while others have shown equivalent TBT outcomes after D5 and D6 cryopreserved blastocysts transfers. STUDY, DESIGN, SIZE, DURATION This retrospective cohort follow-up study included 1347 single autologous frozen-thawed blastocyst transfers performed between January 2012 and December 2015 at a tertiary care university hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS All of the patients scheduled for TBT were allocated to two groups according to the day of blastocyst expansion: on D5 (n = 994) or on D6 (n = 353). The primary outcome was LBR per embryo transfer in the first blastocyst thawing cycle. Secondary outcomes were clinical pregnancy rate (cPR), early miscarriage rate and neonatal outcomes following TBT for the two groups. Statistical analyses were conducted using univariate and multivariate logistic regression model. MAIN RESULTS AND THE ROLE OF CHANCE The LBR was significantly increased in the D5 group compared to the D6 group [294/994 (29.6%) versus 60/353 (17.0%); P < 0.001]. The cPR was also higher when blastocysts were vitrified on D5 compared with those vitrified on D6 [429/994 (43.2%) versus 95/353 (26.9%); P < 0.001]. No significant differences were found between groups in terms of early miscarriage rate (P = 0.862). More good-quality embryos (defined as an B3-B4 or B5 embryo ≥BB according to the grading scale proposed by Gardner) were transferred in the D5 group than in the D6 group [807 (81.2%) versus 214 (60.6%); P < 0.001]. However, a comparison of TBT cycles with equal embryo quality (good versus low) also supported the superiority of D5 blastocysts. Concerning neonatal outcomes, the D5 group infants had a lower mean birth weight compared to those of the D6 group (P = 0.001). In addition, a significantly shorter gestational age at birth is reported in the D5 blastocyst group as compared to the D6 group (P = 0.004). After multivariate logistic regression taking into account potential confounders such as the women's age, number of previous IVF/ICSI procedures, the day of the blastocyst vitrification (D5 or D6) and embryo quality, blastocyst expansion at D6 was independently associated with a significant decrease in LBR compared to D5 expanded-blastocysts (OR 0.52; 95% CI 0.38-0.72; P < 0.001). LIMITATIONS, REASONS FOR CAUTION The poor predictive value of the morphological approach in embryo selection could constitute a limitation in this study. However, blastocyst quality was evaluated similarly in both groups. WIDER IMPLICATIONS OF THE FINDINGS The LBR following frozen-thawed blastocyst transfer was significantly lower with D6 than with D5 blastocysts, regardless of their quality. These results could affect cryopreservation procedures as they suggest that the use of D5-expanded blastocysts for TBT may be preferred in order to shorten the time of conceiving. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was obtained for this study. None of the authors have any competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable.

La spermatogenese est le processus continu de differenciation cellulaire qui aboutit a la production chez l’adulte des spermatozoides. Dans les tubes seminiferes du testicule, ce processus se developpe a partir d’un nombre restreint de cellules, les cellules souches germinales (CSG). Les CSG presentent la double propriete de s’auto-renouveler pour maintenir leur stock, et de se differentier pour produire les cellules hautement specialisees que sont les spermatozoides. L’activite fonctionnelle des CSG chez les mammiferes, dont le primate non-humain, a ete demontree par la regeneration d’une spermatogenese apres leur transplantation dans des testicules depletes en CSG. Chez l’humain, comme chez les autres mammiferes, les CSG sont presentes dans le testicule des la naissance et sont la cible des chimiotherapies et radiotherapies anticancereuses. L’atteinte de leur stock est responsable des sterilites secondaires au traitement. Des biopsies testiculaires sont actuellement realisees chez des patients prepuberes atteints de cancer afin de cryo-conserver les CSG avant l’induction des traitements a haut risque. En effet, l’utilisation potentielle des CSG en therapie cellulaire constitue une alternative interessante pour la restauration de la fertilite masculine. D’apres les modeles animaux, auto-transplantes dans le testicule des patients gueris mais steriles, les CSG permettraient de restaurer la spermatogenese et ainsi une fertilite naturelle ou par assistance medicale a la procreation. Toutefois, l’utilisation des CSG en therapie cellulaire humaine necessite, entre autre, de decrypter les caracteristiques moleculaires et fonctionnelles de ces cellules qui, d’apres les dernieres donnees de la litterature, apparaissent comme une population tres heterogene. Les CSG de souris constituent le modele de CSG le mieux connu chez les mammiferes. Les programmes de recherche, GermPlast et GermCell, menes chez l’humain par nos equipes visent a caracteriser les CSG humaines adultes et prepuberes a partir des donnees acquises chez la souris et a developper des modeles in vivo permettant de mieux comprendre les mecanismes regulant le devenir des CSG humaines en vue de mettre au point les modalites de leur utilisation.

To assess whether high magnification sperm head vacuole examination (SHVE) and/or standard sperm morphology assessment can predict ICSI outcomes in terms of fertilization, embryo quality, and delivery rates, a prospective observational bicentric study was conducted in two publicly funded assisted reproductive technology (ART) units in France between January and July of 2012. A total of 111 ICSI cycles for exclusively male infertility factors were included. A Spearman's correlation test was performed to validate SHVE reproducibility between the ART units. The normal morphology rate and SHVE performed on selected spermatozoa were respectively determined according to David's and Vanderzwalmen's classifications used for motile sperm organelle morphology examination (MSOME) on the day of the ICSI. Receiver Operating Characteristic (ROC) curve analysis was performed to determine thresholds associated with the occurrence of a delivery. There was an excellent correlation between the two operators (r=0.98), thus validating the study's SHVE data. Percentages of normal morphology grade spermatozoa using the standard classification and first-best morphology grade spermatozoa determined by SHVE were not significantly associated with (i) delivery (p=0.58; 0.90 /area under curve (AUC) =0.532; 0.507), (ii) fertilization (p=0.88; 0.90), (iii) top-quality embryos (p=0.27; 0.98), and (iv) good quality embryo rates (p=0.73; 0.98), respectively. In conclusion, high magnification SHVE and standard sperm morphology assessment cannot predict clinical or biological ICSI outcomes.ART: assisted reproductive technology; HBV: hepatitis B virus; HCV: hepatitis C virus; HIV: human immunodeficiency virus; ICSI: intra-cytoplasmic sperm injection; IVF: in vitro fertilization; LNVs: large nuclear vacuoles; MSOME: motile sperm organelle morphology examination; SHVE: sperm head vacuole examination; WHO: World Health Organization.

Microinjection of nuclear vacuole-free spermatozoa selected by motile sperm organellar morphological examination (MSOME) has been claimed to enhance assisted reproduction treatment outcome compared with intracytoplasmic sperm injection. However, the nature of these nuclear vacuoles is unclear, since their localization at the front of the sperm head suggests they might be of acrosomal origin. To study this hypothesis, acrosomal status was evaluated using Pisum sativum agglutinin staining on a smear, together with sperm organellar morphological examination using the same optics as for MSOME on 30 sperm samples from infertile patients, yielding3200 spermatozoa. Vacuoles were present in 61% of spermatozoa when acrosomal material or intact acrosomes were observed, versus 29% when spermatozoa were acrosome reacted (P0.0001). Induction of the acrosomal reaction by ionophore A23587 from 17.4% to 36.1% significantly increased the percentage of vacuole-free spermatozoa from 41.2% to 63.8% (P0.001). These data suggest that most nuclear vacuoles are of acrosomal origin. Hence, the best spermatozoa selected by MSOME are mostly acrosome-reacted spermatozoa. As microinjection of spermatozoa with a persistent acrosome drastically hampers embryo development in animal models, this suggests that the improvement in pregnancy rates reported following intracytoplasmic injection of morphologically selected sperm might be due to the procedure allowing injection of acrosome-reacted spermatozoa.

Objective To evaluate the effects of cyclic QDE peptide (cQDE), derived from sperm fertilin β (ADAM2), in mouse in vitro fertilization (IVF) and its harmlessness on pups after embryo transfer. Design Prospective in vivo and in vitro study in mice. Setting Murine model in an academic research environment in France. Animal(s) Normal B6CBF1 mice. Intervention(s) Indirect immunofluorescence was used to evaluate cQDE binding to oolemma. The IVF assays were run in presence of the species-specific tripeptide (cQDE at 100 μM), and embryos were transferred in pseudopregnant mice. Controls were obtained by natural mating and IVF in regular medium followed by embryo transfer. Main Outcome Measure(s) Evaluation of fertilization, litter size, pup fertility and health. Result(s) Biotinylated cQDE peptide binds to oocyte plasma membrane and increases gamete fusion in cumulus-intact IVF assay. Litter size as well as pup development after embryo transfer showed no statistically significant differences compared with controls. Pups born from embryos that were incubated with the peptide were as healthy and normally fertile as control mice over at least three generations. Conclusion(s) Cyclic QDE is harmless and improves mouse IVF pregnancy rates. A randomized prospective clinical trial to evaluate the beneficial effect of cyclic FEE on human IVF is required before its clinical use.

Des tests d’inhibitions ont permis de proposer l’integrine α6β1 comme le recepteur du spermatozoide sur l’ovocyte mais des experiences d’invalidation de gene ont montre que les sous unites integrines α6 et β1 ovocytaires n’etaient pas essentielles a la fecondation. En utilisant le Western blot et l’immunofluorescence (cytometrie de flux et microscopie), nous avons montre que I’α6β1 est exprimee par les spermatozoides de souris. Comme pour l’ovocyte, un anticorps anti-α6 (GoH3) inhibe specifiquement les capacites fecondantes des spermatozoides.

BACKGROUND Alpha6beta1 integrin has been proposed to act as a sperm receptor on the mouse oocyte by interacting with spermatozoon fertilin beta. We investigated, in humans, whether oocyte integrins could act similarly in gamete fusion, using a cyclic peptide containing the putative disintegrin-binding domain of human fertilin beta [cyclic FEE (cFEE)] and RGD peptide. METHODS Zona-free eggs were inseminated in the absence or presence of peptides. To maintain the membrane protein pattern, the zona pellucida was removed by microdissection. Immunofluorescence and confocal microscopy were used to detect integrin subunits on the oocyte. RESULTS Unexpectedly, cFEE alone increased human gamete fusion by 94% instead of inhibiting fertilization. Furthermore, cFEE together with RGD potentiated the RGD-induced inhibition of fertilization in a dose-dependent manner. The data suggested the hypothesis of integrin cross-talk, further supported by the co-localization of alpha6beta1 and alphavbeta3 integrins, the putative receptors of cFEE and RGD peptides, respectively. CONCLUSIONS RGD-sensitive and -insensitive integrins may be associated in a multimolecular complex working as a sperm receptor on the human oocyte membrane. Supplementation of human IVF culture medium with cFEE peptide might improve fertilization rates in ART.

Objectifs Evaluer le benefice d’une mise en culture prolongee (CP) des embryons de mauvaise qualite (EMQ) par rapport aux embryons de bonne qualite (EBQ). Identifier des facteurs predictifs de grossesse et de naissance apres transfert de blastocystes congeles provenant d’EBQ et d’EMQ. Materiels et methodes Etude de cohorte portant sur l’analyse de 3108 embryons surnumeraires mis en CP apres transfert embryonnaire frais a j2. Mesure des taux de blastulation totale et utile (blastocystes de bonne qualite), de grossesse clinique et de naissance. Analyse des issues neonatales et etude de facteurs predictifs de grossesse et de naissance apres transfert de blastocystes congeles. Resultats Le taux de blastulation totale etait de 48,7 % a partir d’EMQ et de 67,2 % a partir d’EBQ. Le pourcentage de blastocystes de bonne qualite etait respectivement de 47,9 % et 60,7 % incluant 7,3 % et 14,7 % de blastocystes de top-qualite. Au total, 150 blastocystes provenant d’EBQ et 729 d’EMQ ont ete cryopreserves. A partir de ces blastocystes congeles, 37 et 161 ont ete decongeles et transferes respectivement obtenant 19 (51,4 %) et 61 (37,9 %) grossesses cliniques. Ces dernieres ont abouti a 13 (35,1 %) naissances dans le groupe d’EBQ et 32 (19,9 %) naissances dans le groupe d’EMQ (p = 0,046). Fait interessant, la qualite des blastocystes a l’origine des naissances etait equivalente dans les deux groupes. Parallelement, aucune difference n’a ete mise en evidence quant aux issues neonatales. Apres analyse multivariee, l’âge feminin Conclusion Les EMQ sont capables d’atteindre le stade de blastocyste, de s’implanter et d’etre a l’origine de naissances aux issues neonatales equivalentes a celles observees a partir des EBQ. L’âge feminin

International audience; Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

Cell fusion occurs when cells unit their membranes and share their ­cytoplasm. Cell fusion may occur between genetically identical or different cells. Gamete fusion is a short event of the organism life cycle but it is essential for all organisms that depend on sexual reproduction for the maintenance of the species. The fusion of gametes having inherited one half of the genetic material of their respective parents ensures the diversity of individuals within a population. This diversity is increased by the exchange of genetic material between homologous chromosomes during meiosis. Therefore gamete fusion is a critical biological step in the life cycle that has recently raised more interest due to the possibility of treating patients with a fertility defect by in vitro methods. Molecular mechanisms underlying the fusion process are far from evident since (1) gene-knock out technologies developed to test in vivo the data from in vitro fertilization (IVF) studies failed to confirm the essential role of most of previously candidate molecules and (2) the surface proteins involved are deprived of fusogen properties. Indeed, among the large number of in vivo tested proteins, only CD9 and CD81 tetraspanins on egg and Izumo on sperm have been shown to be essential in mammalian sperm-egg membrane fusion and none of these molecules contain a fusion peptide.

But de l’etude Dans les pays industrialises, on estime qu’un couple sur six consulte pour des problemes de fertilite. L’etat actuel des connaissances chez l’animal permet d’envisager le developpement de techniques de therapie cellulaire utilisant les cellules souches germinales (CSG), qui assurent le maintien de la spermatogenese dans le testicule, dans le but de restaurer une fertilite apres transplantation testiculaire. Notre projet vise a caracteriser les CSG humaines. Methodes Des suspensions cellulaires preparees a partir de biopsies testiculaires humaines ont ete analysees par cytometrie en flux a l’aide du fluorochrome specifique de l’ADN Vybrant pour le marqueur side population (SP) et des marqueurs α6 integrine, Thy-1, et β-2 microglobuline. Pour l’etude fonctionnelle des CSG humaines, nous avons developpe un test in vivo apres transplantation des cellules dans les testicules de souris immunodeficientes ‹‹ humanisees ›› NSG, permettant d’observer un debut de colonisation des tubules seminiferes murins par les CSG humaines. Resultats Ces marqueurs permettent de definir differentes sous-populations germinales, dont une sous-population SPα6 + Thy-1 + β2M qui contient les progeniteurs spermatogoniaux les plus immatures. Le test de fonctionnalite apres transplantation montre que les CSG sont presentes dans la sous-population cellulaire SPα6 + Thy-1 + β2 M−. D’autre part, differentes conditions de culture in vitro ont ete testees et permettent la culture des cellules souches ‹‹ spermatogonies immatures ›› sur une periode de 21 jours. Conclusion Nous avons ainsi developpe une methode permettant d’etudier la differenciation germinale au cours de la spermatogenese adulte humaine, et de purifier une population hautement enrichie en GSG.

Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis ofin vitroexchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists inCd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process.

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm–egg fusion is a direct consequence of CD9 controlled sperm–egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.

To report the level of leukocytospermia in fertile donors' semen. Surprisingly, seminal leukocytes protect fertilization properties of sperm and are associated with normal or improved assisted reproductive technology outcomes in infertility patients. This raises the question of whether leukocytospermia exists in fertile men as well. We report a study of sperm donors who, by law in France, have to be of proven fertility.Retrospective analysis.University laboratory.One hundred fifty-five donors were selected for cryobanking. Results of their sperm analyses were compared with those from 10,242 infertile men.None.The men's first ejaculate was studied by cytologic analysis to determine the round cell and polymorphonuclear cell (PMN) contents. A total of 3,875 donor sperm inseminations (DSIs) were performed, and their outcomes were analyzed over an 8-year period.PMN is more elevated in semen from infertility patients than in semen from fertile donors, but some donors (6.5%) had high leukocytospermia (≥10(6)/mL). The post-DSI pregnancy rate was increased when round cells were present (P.02) but not with higher PMN concentrations. Furthermore, high leukocytospermia was associated with an increased post-DSI miscarriage rate.In fertile donors, as in infertility patients, high leukocytospermia (10(6)/mL) is associated with a normal pregnancy rate but an increased percentage of early pregnancy loss.

L’ovocyte qui va etre feconde a grandi en taille au sein de son follicule et s’est entoure de la zone pellucide, structure amorphe acellulaire composee de glycoproteines au nombre de quatre dans l’espece humaine (1). Cette zone pellucide va avoir plusieurs fonctions: la reconnaissance et la selection du spermatozoide fecondant, ainsi que le declenchement de sa reaction acrosomique lui permettant de liberer les enzymes qu’il transporte dans l’acrosome. Ceux-ci vont lui permettre de rompre les liaisons entre la ZP1 et les polymeres de ZP2-ZP3 afi n de se frayer un passage et de se propulser au travers de la zone pellucide grâce a ses battements flagellaires jusque dans l’espace perivitellin (2).

Background Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. Results Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process. Conclusion These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported.

Several families of molecules are implicated in the membrane fusion process between sperm and oocyte. Among these, CD9 tetraspanin, a membrane-organizing molecule, plays a crucial role, since the fertilizing ability of CD9-/- oocytes is dramatically impaired. CD9 controls alpha6-beta1 integrin relocation involved in the membrane reorganization that occurs on oocyte fertilization but is not expressed on sperm. We report here that, together with several other proteins, the CD9 tetraspanin is transferred from the oocyte to the fertilizing spermatozoa present in the perivitelline space before fertilization. Transfer of CD9 from oocyte to sperm from CD9-/- male mice still occurs. CD9 acquisition by sperm results from a transfer of membrane fragments from the plasma membrane of the oocyte, in a process similar to trogocytosis, the recently described mechanism of intercellular exchange of membrane patches. Acquisition of CD9 by the sperm may be crucial for the membrane reorganization in sperm required for fusion with the oocyte, a process that is similar to the role CD9 plays in oocyte membrane reorganization.

The feasibility of using spermatogonial stem cells (SSCs) for cell-based infertility treatment has suffered from a lack of evidence in a preclinical nonhuman primate model. In this issue, Hermann et al. (2012) demonstrate that autologous and allogeneic transplantation of SSCs in testes of rhesus macaques produced functional sperm.