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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.

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.

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.

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.

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.

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.

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.

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.