Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

Efficacy of assisted reproductive technology after ovarian tissue transplantation in a cohort of 11 patients with or without associated infertility factors.

PURPOSE: IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor reproductive outcomes. The aim of this study was to evaluate the efficacy of ovarian tissue transplantation (OTT) followed by assisted reproductive technology (ART) in women with or without associated infertility factors. METHODS: This is a prospective cohort study with retrospective data collection including eleven women, four of whom having an infertility factor (IF), who had undergone OTT in one university center between 2005 and 2017, followed by ART in six in vitro fertilization (IVF) centers. RESULTS: In total, 25 of the 85 cycles initiated (29%) were canceled, resulting in 60 oocyte retrievals. Ninety-five oocytes were retrieved: 36 were abnormal or immature, 29/39 fertilized (74%) after ICSI and 13/20 (65%) after IVF. Thirty-five embryos were transferred in seven patients (5/7 patients without IF and 2/4 patients with IF). After ART, one patient with IF experienced two pregnancies, one resulting in a live birth. For all patients, pregnancy rates and live birth rates were 7.4% and 3.7% per embryo transfer, respectively. Nine pregnancies and four live births occurred after spontaneous conception in five patients without IF, none in the infertility group. CONCLUSION: This study confirms that IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor outcomes. However, the chances of natural conception are high in women without IF. Patients with IF, without the possibility of spontaneous pregnancy, should be informed of poor reproductive outcomes after OTT followed by ART. TRIAL REGISTRATION NUMBER: NCT02184806.

JUNO, the receptor of sperm IZUMO1, is expressed by the human oocyte and is essential for human fertilisation.

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'Immunogénétique (ICIG). There are no competing interests.

[Prostatitis and fertility: the biologist's point of view]. / Prostatite, fertilité : le point de vue du biologiste.

There are two leukocytospermia: the leukocytospermia below 10(6) cells/ml comes from the epididymis. It is physiological, improves sperm quality and ART outcomes and therefore must be respected. Leukocytospermia above 10(6) cells/ml are of prostatic origin and reflect a chronic prostatitis. The results of IVF and ICSI with these sperm are always surprisingly improved when compared to those obtained using semen without leukocytes at all. But this improvement is offset by a dramatic increase in the miscarriage rate. Should we treat this leukocytospermia or its cause? A clinical trial is conducted in Cochin hospital with the PHRC Sigma (Male Genital Track Inflammatory Syndrome) that will help us answer this question. It seems, a priori, that it is better to treat the cause and to respect the leukospermia.

Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin.

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, yielding >3200 spermatozoa. Vacuoles were present in 61% of spermatozoa when acrosomal material or intact acrosomes were observed, versus 29% when spermatozoa were acrosome reacted (P<0.0001). 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% (P<0.001). 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.

Short gamete co-incubation during in vitro fertilization decreases the fertilization rate and does not improve embryo quality: a prospective auto controlled study.

PURPOSE: Evaluate the effect of short gamete incubation on fertilization rate and embryo quality. METHODS: A prospective study has been performed. Two thousand five hundred and forty seven sibling oocytes from 240 couples undergoing IVF attempts were allocated to a short (1 h) or a standard (18 h) insemination procedure. Diploid fertilization rate (two pronuclei, 2PN), polyspermy (>2PN) and embryo quality were compared. RESULTS: The fertilization rate was statistically lower in the short insemination group compared to the standard insemination one (64.9% and 70.1%; P = 0.039), with a similar polyspermy rate observed between the two groups. A slight, but non significant, increase was observed concerning good embryo quality rate in the short insemination group when compared to the standard insemination, both at day 2 (60.1 vs. 58.1%; P = 0.06) and day 3 (53.2 vs. 48.5%; P = 0.22). CONCLUSION: This new study highlights that a 1 h gamete exposure decreases the fertilization rate and does not improve embryo quality compared with a standard 18 h insemination procedure.

Cyclic FEE peptide increases human gamete fusion and potentiates its RGD-induced inhibition.

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.

[Leucocytospermia, oxidative stress and male fertility: facts and hypotheses]. / Leucospermie, stress oxydatif et fertilité masculine: certitudes et hypothèses.

Leukocytospermia is frequent and significantly increased (over 10(6)/ml) in 20% of male factor infertility. It induces the production of highly toxic reactive oxygen species (ROS) which impair genital track accessory glands and sperm cell functions. The seminal medium contains extremely potent antioxidative defenses which usually balance the oxidative stress. In vivo, these defenses can be overwhelmed when ROS production is extremely important and/or when it lasts for a very long period of time. Infertility can then appear. In vitro, ROS have been univoqually demonstrated for being highly toxic since spermatozoa are no longer protected. Sperm cell defects are : decrease of acrosome reaction and fusiogenic ability and increase of DNA fragmentations. In case of male factor infertility, a leukocytospermia represents an essential or an additional risk factor that should be treated, specially when in vitro therapy is to be scheduled, in order to improve gamete quality.