Mechanics of human embryo compaction.

The shaping of human embryos begins with compaction, during which cells come into close contact1,2. Assisted reproductive technology studies indicate that human embryos fail compaction primarily because of defective adhesion3,4. On the basis of our current understanding of animal morphogenesis5,6, other morphogenetic engines, such as cell contractility, could be involved in shaping human embryos. 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 shows a fourfold increase of tension at the cell-medium interface whereas cell-cell contacts keep a steady tension. Therefore, increased tension at the cell-medium interface drives human embryo compaction, which is qualitatively similar to compaction in mouse embryos7. Further comparison between human and mouse shows 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 shows that, whereas both cellular processes are required for compaction, only contractility controls the surface tensions responsible for compaction. Cell contractility and cell-cell adhesion exhibit distinct mechanical signatures when faulty. Analysing the mechanical signature of naturally failing embryos, we find evidence that non-compacting or partially compacting embryos containing excluded cells have defective contractility. Together, our study shows that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.

A 20-year overview of fertility preservation in boys: new insights gained through a comprehensive international survey.

STUDY QUESTION: Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER: Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY: For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN SIZE DURATION: A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS SETTING METHODS: Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6-20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS REASONS FOR CAUTION: While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS: Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTERESTS: K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation-Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children's Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G., N.N., S.S., C.L.M., A.M.M.v.P., C.E., R.T.M., K.D., M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Ovarian tissue cryopreservation for fertility preservation before hematopoietic stem cell transplantation in patients with sickle cell disease: safety, ovarian function follow-up, and results of ovarian tissue transplantation.

PURPOSE: To describe the experience of performing ovarian tissue cryopreservation (OTC) before hematopoietic stem cell transplantation (HSCT), among girls/women with severe sickle cell disease (SCD)(SS or S/ß0-thalassemia) who are, besides the usual surgical risk, at risk of SCD-related complications during the fertility preservation procedure for improving their counseling and management. METHODS: This retrospective study included 75 patients (girls/women) with SCD who have had OTC before myeloablative conditioning regimen (MAC) for HSCT. Characteristics of patients and data on OTC, ovarian status follow-up, and results of ovarian tissue transplantation (OTT) were collected in medical records. RESULTS: At OTC, the median (IQR 25-75; range) age of the patients was 9.6 (6.9-14.1; 3.6-28.3) years, 56/75 were prepubertal, and no SCD or surgery-related complications occurred. The median follow-up post-HSCT was > 9 years. At the last follow-up, among prepubertal patients at HSCT, 26/56 were ≥ 15 years old and presented with a premature ovarian insufficiency (POI), except 2, including the patient who had received an OTT to induce puberty. Eight were 13-15 years old and presented for POI. The remaining 22 patients were under 13. Among the 19 patients who were menarche at HSCT, 2 died 6 months post-HSCT and we do not have ovarian function follow-up for the other 2 patients. All the remaining patients (n = 15) had POI. Five patients had OTT. All had a return of ovarian function. One patient gave birth to a healthy baby. CONCLUSION: OTC is a safe fertility preservation technique and could be offered before MAC independent of the patient's age.

A 10-year experience in testicular tissue cryopreservation for boys under 18 years of age: What can be learned from 350 cases?

BACKGROUND: A growing number of centers worldwide are preserving testicular tissue (TT) of young boys at risk of fertility loss to preserve their fertility. Data in this regard are scarce and experience sharing is essential to the optimization of the process. OBJECTIVES: This report of our 10-year activity of pediatric fertility preservation (FP) has the objective to (1) improve knowledge regarding the feasibility, acceptability, safety, and potential usefulness of the procedure; (2) analyze the impact of chemotherapy on spermatogonia in the cryopreserved TT. MATERIALS AND METHODS: For this retrospective study of data prospectively recorded, we included all boys under 18 years of age referred to the FP consultation of our academic network between October 2009 and December 2019. Characteristics of patients and cryopreservation of testicular tissue (CTT) were extracted from the clinical database. Univariate and multivariate analyses were used to assess factors associated with the risk of absence of spermatogonia in the TT. RESULTS: Three hundred and sixty-nine patients (7.2 years; 0.5-17.0) were referred to the FP consultation for malignant (70%) or non-malignant (30%) disease, of whom 88% were candidates for CTT, after a previous chemotherapy exposure (78%). The rate of recorded immediate adverse events was 3.5%, with painful episodes dominating. Spermatogonia were detected in the majority of TTs: 91.1% of those exposed to chemotherapy and 92.3% of those not exposed (p = 0.962). In multivariate analysis, the risk of absence of spermatogonia was almost three-fold higher in boys > 10 years of age ([OR] 2.74, 95% CI 1.09-7.26, p = 0.035) and four-fold higher in boys exposed to alkylating agents prior to CTT ([OR] 4.09, 95% CI 1.32-17.94, p = 0.028). DISCUSSION/CONCLUSION: This large series of pediatric FP shows that this procedure is well accepted, feasible, and safe in the short term, strengthening its place in the clinical care pathway of young patients requiring a highly gonadotoxic treatment. Our results demonstrate that CTT post-chemotherapy does not impair the chance to preserve spermatogonia in the TT except when the treatment includes alkylating agents. More data on post-CTT follow-up are still required to ensure the long-term safety and usefulness of the procedure.

What should be done in terms of fertility preservation for patients with cancer? The French 2021 guidelines.

AIM: To provide practice guidelines about fertility preservation (FP) in oncology. METHODS: We selected 400 articles after a PubMed review of the literature (1987-2019). 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 first-line 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 is to 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.

Transcriptional profiling of ß-2M-SPα-6+THY1+ spermatogonial stem cells in human spermatogenesis.

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 ß-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype ß-2 microglobulin (ß-2M)-SPα-6+THY1+, which is highly enriched in stem cells. By analyzing the expression signatures of this SSC-enriched population along with other germinal progenitors, we established an exhaustive transcriptome of human spermatogenesis. Transcriptome profiling of the human ß-2M-SPα-6+THY1+ population and comparison with the profile of mouse undifferentiated spermatogonia provide insights into the molecular networks and key transcriptional regulators regulating human SSCs, including the basic-helix-loop-helix (bHLH) transcriptional repressor HES1, which we show to be implicated in maintenance of SSCs in vitro.

Case Report: Abnormalities of sperm motility and morphology in a patient with Leber hereditary optic neuropathy: Improvement after idebenone therapy.

Case: We report the sperm characteristics of a male patient who developed, when he was 18 years old, a Leber hereditary optic neuropathy, a hereditary optic neuropathy due to mtDNA mutation as well as variants in the nuclear DNA. At the age of 30 years-old, he complained of infertility lasting for 2 years. Semen analyses showed low motility spermatozoa and a high percentage of morphological or ultrastructural abnormalities. Levels of epididymal markers were strongly atypical. Idebenone was prescribed as treatment of his Leber hereditary optic neuropathy in order to improve his visual acuity. After 5 months of this treatment, motility of spermatozoa increased, and their vitality improved. A natural conception occurred. Outcome: This case is the first description of an anomaly of spermatozoas and of the epididymis epithelium in a patient with Leber hereditary optic neuropathy. It draws attention to sperm pathologies in patients with mitochondrial disorders. The role of the mtDNA mutations must be suspected since it plays an important role in the development and motility of spermatozoa. In addition, idebenone can by-pass the complex I and transfer electrons to complex III. It has been suspected to have a favorable effect on spermatogenesis. Conclusion: This case confirms the possibility of sperm dysfunction in Leber hereditary optic neuropathy and the interest of idebenone as a treatment for infertility due to mtDNA mutations in human.

Partial Sperm beta1 Integrin Subunit Deletion Proves its Involvement in Mouse Gamete Adhesion/Fusion.

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.

Contribution of Single-Cell Transcriptomics to the Characterization of Human Spermatogonial Stem Cells: Toward an Application in Male Fertility Regenerative Medicine?

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.

Live birth rate following frozen-thawed blastocyst transfer is higher with blastocysts expanded on Day 5 than on Day 6.

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.

Extended culture of poor-quality supernumerary embryos improves ART outcomes.

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.

Predicting the clinical outcome of ICSI by sperm head vacuole examination.

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. ABBREVIATIONS: 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.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

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.

Stem cell therapy for male infertility takes a step forward.

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.

Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes.

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 of in vitro exchanges 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 in Cd9-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.

Seminal leukocytes are Good Samaritans for spermatozoa.

OBJECTIVE: To assess the effect of leukocytospermia on assisted reproductive technology outcomes. DESIGN: Retrospective analysis. SETTING: University laboratory. PATIENT(S): Couples attending the infertiliy clinic and involved in ART program for IVF or ICSI. INTERVENTION(S): During a 7-year follow-up in an assisted reproductive technology program, leukocytospermia was routinely determined using the peroxidase technique. Donor sperm were excluded from the study. MAIN OUTCOME MEASURE(S): Egg retrievals (N = 3,508) were distributed in 3 groups according to the leukocyte levels in semen from which fertilizing sperm were extracted: group 1, absence of leukocytes (n = 3,026); group 2, moderate leukocytospermia (<10(6)/mL) (n = 344); or group 3, high leukocytospermia (≥10(6)/mL) (n = 138). They resulted in 1,463 IVF and 2,045 intracytoplasmic sperm injection procedures that gave 802 clinical pregnancies. RESULT(S): Surprisingly, the fertilization rate, cleavage rate, clinical pregnancy rate, gestational age, and mean infant weight were significantly improved when seminal leukocytes were present, regardless of the technique used. The only negative side effects associated with a high level of seminal leukocytes (group 3) were an elevated rate of early pregnancy loss (from 26.6% to 40.5%) and a 3-fold increase in the percentage of ectopic pregnancies. CONCLUSION(S): At moderate levels (<10(6)/mL), leukocytospermia appears to be physiologic. It is associated with improved sperm fertilization ability and pregnancy outcome. At higher concentrations, leukocytospermia alters neither sperm fertilization ability nor the probability of clinical pregnancy when compared with nonleukocytic patients with infertility. However, the pregnancy outcome is reduced.

Seminal leukocytes and clinical outcomes with donor sperm insemination.

OBJECTIVE: 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. DESIGN: Retrospective analysis. SETTING: University laboratory. PATIENT(S): One hundred fifty-five donors were selected for cryobanking. Results of their sperm analyses were compared with those from 10,242 infertile men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 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. RESULT(S): 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. CONCLUSION(S): 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.

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization.

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.