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Resume Contexte Ces recommandations communes de l’Association Francaise d’Urologie (AFU) et de la Societe d’Andrologie de Langue Francaise (SALF) ont pour objectif de fournir des directives pratiques a la communaute urologique et andrologique Francaise concernant l’evaluation des hommes infertiles. Materiel et methodes Recherche bibliographique dans PubMed en utilisant les mots-cles «male infertility», «diagnosis», «management» et «evaluation» limitee aux articles cliniques en anglais et en francais anterieurs au 1/01/2020. Pour renseigner le niveau de preuves, le systeme de gradation de l’HAS (2013) a ete applique. Resultats Concernant l’evaluation des hommes infertiles, l’AFU et la SALF recommandent : (1) un interrogatoire systematique explorant les antecedents familiaux, les antecedents de fertilite de l’homme hors couple, les antecedents personnels du patient pouvant avoir un impact sur sa fertilite, ses habitudes de vie, ses traitements, ses symptomes et les eventuelles difficultes sexuelles du couple ; (2) un examen physique general pour evaluer les signes d’hypogonadisme et les caracteres sexuels secondaires ; (3) un examen physique scrotal par un urologue ou un andrologue pour evaluer (i) le volume et la consistance des testicules, (ii) les canaux deferents et les epididymes a la recherche d’une absence totale ou partielle ou des nodules, et (iii) la presence de varicocele ; (4) d’effectuer 2 spermogrammes, conformement aux recommandations de l’Organisation mondiale de la sante (OMS), si le premier presente au moins une anomalie ; (5) une echographie scrotale systematique, pouvant etre completee par une echographie pelvienne endorectale selon la clinique ; (6) un bilan hormonal comportant au moins une determination de la testosterone serique et de FSH ; si la testosterone est basse, la LH permettra de differencier hypogonadisme hypogonadotrope (central) et hypogonadisme peripherique ; (7) un caryotype chez les hommes infertiles ayant une concentration de spermatozoides ≤ 10,106/ml ; (8) l’evaluation des microdeletions du chromosome Y chez les hommes infertiles ayant une concentration de spermatozoides ≤ 1,106/ml ; (9) l’evaluation du gene CFTR en cas de suspicion d’agenesie bilaterale ou unilaterale des canaux deferents et des vesicules seminales. La place de l’analyse de l’integrite de l’ADN du sperme (SCSA, TUNEL par exemple) dans le bilan de l’homme infertile est encore en cours d’evaluation. Conclusion Ces recommandations peuvent etre appliquees en pratique clinique en routine chez tout homme infertile.

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.

Germline mosaicism is considered to be a rare event. However, its occurrence is underestimated due to the limited availability of germ cells. The genomic variations that underlie this phenomenon comprise single nucleotide polymorphism (SNPs), copy number variations (CNVs) and aneuploidies. In the case of CNVs, deletions are more frequent in the paternal germline while duplications are more commonly maternal in origin. Germline mosaicism increases with paternal age as the risk of SNPs increase with the number of germ cell divisions. We here report a case of germline mosaicism in the spermatozoa of a donor that resulted in one pathological pregnancy.Straws from the same sperm donor were provided to seven recipient couples, resulting in four pregnancies. Second trimester ultrasound analysis revealed bilateral talipes equinovarus associated with growth retardation in one of these pregnancies. Array-comparative genomic hybridization (CGH) carried out after amniocentesis revealed a 4 Mb deletion in the 7q32.1q33 region. The blood karyotypes and array-CGHs were normal in the mother, as well as in the donor. However, the microsatellite profile indicated a paternal origin. Fluorescent in situ hybridization (FISH) analysis of the donor's spermatozoa revealed the same chromosomal rearrangements in 12% of the spermatozoa population. Due to the documented risk of mental retardation associated with genomic rearrangements in the same region, the couple decided to terminate the pregnancy. Amniocentesis was performed in the other couples, which yielded normal FISH analysis results.Several cases of germline mosaicism have been reported to date, but their frequency is probably underestimated. Moreover, it is important to note that germline mosaicism cannot be ruled out by conventional cytogenetic screening of blood cells. This case highlights the need for close follow-up of every pregnancy obtained through gamete donation, given that the occurrence of germline mosaicism may have major consequences when multiple pregnancies are obtained concomitantly.La mise en évidence d’une mosaïque germinale est. un événement rare mais probablement sous-estimé du fait de l’accès limité aux cellules germinales. Les variations génomiques caractéristiques de ce phénomène peuvent être des single nucleotide polymorphismes (SNPs), des copy number variations (CNVs) ou des aneuploïdies. Dans le cas des CNVs, les délétions sont plus fréquentes dans la lignée germinale paternelle tandis que les duplications sont plus fréquemment d’origine maternelle. Le risque de mosaïcisme germinal augmente avec l’âge paternel de part une augmentation du risque de SNPs associée à la division constante des cellules germinales pendant toute la vie d’un homme. Nous rapportons ici un cas de mosaïque germinale chez un donneur de spermatozoïdes ayant entraîné la survenue d’une grossesse pathologique.Les paillettes d’un même donneur de spermatozoïdes ont été attribuées à sept couples receveurs permettant l’obtention de quatre grossesses évolutives. Pour l’une d’entre elle, l’échographie du deuxième trimestre a permis d’identifier chez le fœtus des pieds bots associés à un retard de croissance intra utérin. L’analyse par hybridation génomique comparative (CGH)-array après amniocentèse a révélé une délétion de 4 MB dans la région 7q32.1q33. Les caryotypes sanguins et les analyses par CGH-array étaient normaux chez la mère et le donneur. Cependant les profils de microsatellites ont montré une origine paternelle du chromosome délété. Une analyse par fluorescent in situ hybridization (FISH) des spermatozoïdes du donneur a révélé la présence de la même délétion dans 12% des spermatozoïdes étudiés. Etant donné le risque de retard mental associé à des remaniements chromosomiques dans cette même région, le couple a préféré interrompre la grossesse. Une amniocentèse a été réalisée pour les autres grossesse en cours et n’a retrouvé aucune anomalie.Plusieurs cas de mosaïques germinales ont été rapportés mais leur fréquence réelle reste probablement sous-estimée. En effet, un mosaïcisme germinal ne peut être détecté par les techniques de cytogénétique conventionnelle sur sang. Ce cas illustre la nécessité d’un suivi en temps réel des grossesses obtenues par don de spermatozoïdes étant donné que la survenue d’une grossesse pathologique peut avoir un retentissement sur les autres grossesses issues du même donneur.

BACKGROUND More than half of transgender (TG) men wish to have children. Until recently, TG people in France were rarely offered gamete donation, mainly because the Bioethics Law allows the use of assisted reproductive technologies (ART) only in infertile couples. The only option currently available for heterosexual couples with a TG man is ART with sperm donation. The Center for Study and Preservation of Eggs and Sperm (CECOS) of the Cochin Hospital is the first French center to propose sperm donation to such couples, and has done so since 1999. OBJECTIVES To determine the main characteristics and intentions of 43 couples, including a TG man and his cisgender female partner awaiting sperm donation. MATERIALS AND METHODS A retrospective analysis was carried out on the records from October 2010 to December 2019, of 43 couples with a TG man who applied for sperm donation at the CECOS of the Cochin Hospital (Paris, France). RESULTS The mean age of TG men and cisgender women was 32 ± 6.6 and 29.7 ± 4.6 years, respectively. In 77% of cases, the couple met before the man's transition. Eighty-one percent of the couples were in a stable relationship for at least 3 years, and 94% wished to have a child for no more than 5 years. Almost all of the couples (95%) intended to inform their child of their conception by sperm donation and the father's transidentity (92%). DISCUSSION Due to restrictive French legislation, the profile of our couples probably does not reflect that of all couples consisting of a TG man and a cisgender woman. The study took place over a long period of time and the characteristics of the couples could probably change over time. CONCLUSION The couples often met before the man's transition, cohabited for several years, intended to inform their child of sperm donation and the father's transidentity.

Background Spinal cord injury often results in erectile dysfunction and an ejaculation along with impaired semen parameters. Fertility is a major concern in spinal cord injury adult males and some fear that the delay post-spinal cord injury may negatively affect sperm quality. Objectives We aimed to (i) assess semen parameters over time in SCI patients according to age at spinal cord injury, time post-spinal cord injury, and the spinal cord injury level and completeness and (ii) measure markers in semen for inflammation and marker of oxidative stress to investigate their impact on sperm parameters. Materials and methods The study is a prospective, longitudinal, pilot study over 18 months. Thirty-five men with spinal cord injury from 18 to 60 years of age were enrolled. Their mean age was 29.4 ± 6.4 years. Semen retrieval was scheduled every 6 months, allowing analysis of four ejaculates, in association with measurement of granulocyte and seminal plasma elastase concentrations to assess markers in semen for inflammation and spermatozoa DNA fragmentation to assess oxidative stress. Results Based on reference limits, a normal total sperm number, decreased motility and vitality of the spermatozoa, and increased morphological abnormalities were found. Mean round cell and granulocyte concentrations were elevated in the semen. Markers in semen for inflammation and marker of oxidative stress were elevated in several semen samples, compared to reference limits. However, neither the presence of markers in semen for inflammation or oxidative stress, the completeness or the level of the spinal cord lesion, the age or the time post-spinal cord injury had a negative impact on the semen quality over time. Discussion There was no significant decline in semen quality in spinal cord injury patients over time within the limitations of this pilot study. Moreover, a chronic genital inflammatory status was not associated with impairment of semen quality. Conclusion The present findings are reassuring for men with spinal cord injury and could guide the management of their reproductive ability. According to these preliminary data, not all spinal cord injury patients who are able to ejaculate require systematic freezing of their spermatozoa.

STUDY QUESTION How efficacious is transplantation of ovarian cortex previously exposed to chemotherapy? SUMMARY ANSWER Prior exposure to chemotherapy did not disrupt the function of cryopreserved ovarian tissue after transplantation. WHAT IS KNOWN ALREADY Ovarian tissue cryopreservation (OTC) followed by ovarian tissue transplantation (OTT) is an efficacious technique for restoration of female fertility. At least 130 children have been born following this procedure. To date, little is known about the efficacy of OTT in patients exposed to cancer chemotherapy prior to OTC. STUDY DESIGN, SIZE, DURATION This study evaluates the recovery of ovarian function and fertility in 31 consecutive patients who had received OTT, between 2005 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS Thirty one patients, wanting children, were transplanted with autologous ovarian cortex, among which 22 patients (71%) had been exposed to chemotherapy before OTC. Recovery of ovarian function was considered total once menstruation occurred. Ovarian function recovery (OFR), ovarian graft survival, and incidence of pregnancy were related to previous chemotherapy exposure, type of chemotherapy and graft characteristics (number of grafted fragments and follicular density). MAIN RESULTS AND ROLE OF CHANCE The amount of ovarian tissue collected was the only parameter to show any significant change between patients with versus without previous chemotherapy. At 1 year after OTT, the cumulative incidence of OFR was 83% (93% in patients exposed to chemotherapy and 67% in others (P = 0.14)). A low follicular density (

Introduction The preservation of fertility is an integral part of care of children requiring gonadotoxic treatments for cancer or non-malignant diseases. In France, the cryopreservation of ovarian tissue has been considered and has been offered as a clinical treatment since its inception. The aim of this study is to review 20 years of activity in fertility preservation by ovarian tissue cryopreservation (OTC) for children and the feasibility of oocyte isolation and cryopreservation from the ovarian tissue at a single center. Material and methods Retrospective study including patients aged 15 years or younger who underwent OTC, combined for some with oocyte cryopreservation of isolated oocytes, before a highly gonadotoxic treatment for malignant or non-malignant disease was initiated. We describe the evolution of activities in our program for fertility preservation and patient characteristics at the time of OTC and follow up. Results From April 1998 to December 2018, 418 girls and adolescents younger than 15 years of age underwent OTC, representing 40.5% of all females who have had ovarian tissue cryopreserved at our center. In all, 313 patients had malignant diseases and 105 had benign conditions. Between November 2009 and July 2013, oocytes were isolated and also cryopreserved in 50 cases. The mean age of patients was 6.9 years (range 0.3-15). The most frequent diagnoses in this cohort included neuroblastoma, acute leukemia and hemoglobinopathies; neuroblastoma being the most common diagnosis in very young patients. During follow up, three patients requested the use of their cryopreserved ovarian tissue. All had undergone ovarian tissue transplantation, one for puberty induction and the two others for restoring fertility. So far, no pregnancies have been achieved. Eighty-four patients who had OTC died. Conclusions Ovarian tissue cryopreservation is the only available technique for preserving fertility of girls. To our knowledge this is the largest series of girls and adolescents younger than 15 years so far reported on procedures of OTC before highly gonadotoxic treatment in a single center.

International audience; Background. — The aim of these Association Franc ̧aise d’Urologie (AFU) and Sociétéd’Andrologie de Langue Franc ̧aise (SALF) common recommendations are to provide practiceguidelines for the French Urological and Andrological community regarding the evaluation ofinfertile men.Material and methods. — Literature search in PubMed using the keywords ‘‘male infertility’’, ‘‘diagnosis’’, ‘‘management’’ and ‘‘evaluation’’ limited to clinical articles in English and French prior to 01/01/2020. To inform the level of evidence, the HAS grading system (2013) was applied.Results. — Concerning the evaluation of infertile men, the AFU and the SALF recommend : (1)a systematic interview exploring the family history, the fertility history of the man outside thecouple, the patient’s personal history that may have an impact on his fertility, lifestyle habits,treatments, symptoms and possible sexual difficulties of the couple; (2) a general physical examination to assess signs of hypogonadism and secondary sexual characters; (3) a scrotal physical examination performed by an urologist or andrologist to assess (i) the testes for volume and consistency, (ii) vas deferens and epididymes for total or partial absence or nodules, and(iii) presence of varicoceles; (4) Performing two semen analyses, according to World HealthOrganization guidelines, if the first one has at least one abnormaly; (5) a scrotal ultrasoundas part of routine investigation, that can be completed with an endorectal pelvic ultrasound according to the clinic; (6) an endocrine evaluation with at least a Testosterone and FSH serum determination; (7) Karyotype analysis in infertile men with a sperm concentration ≤ 10 106/mL;(8) assessment of Yq microdeletions in infertile men with a sperm concentration ≤ 1 106/mL;(9) Cystic fibrosis trans membrane conductance regulator gene evaluation in case of suspicion for bilateral or unilateral congenital agenesis of vas deferens and seminal vesicles.; Contexte. — Ces recommandations communes de l’Association Française d’Urologie (AFU) et de la Société d’Andrologie de Langue Franc ̧aise (SALF) ont pour objectif de fournir des directives pratiques à la communauté urologique et andrologique Française concernant l’évaluation des hommes infertiles.Matériel et méthodes. —Recherche bibliographique dans PubMed en utilisant les mots-clés «male infertility», «diagnosis», «management» et «evaluation» limitée aux articles cliniques en anglais et en français antérieurs au 01/01/2020. Pour renseigner le niveau de preuves, le système de gradation de l’HAS (2013) a été appliqué.Résultats. — Concernant l’évaluation des hommes infertiles, l’AFU et la SALF recommandent :(1) un interrogatoire systématique explorant les antécédents familiaux, les antécédents de fertilité de l’homme hors couple, les antécédents personnels du patient pouvant avoir un impact sur sa fertilité, ses habitudes de vie, ses traitements, ses symptômes et les éventuelles difficultés sexuelles du couple ; (2) un examen physique général pour évaluer les signes d’hypogonadisme et les caractères sexuels secondaires ; (3) un examen physique scrotal par un urologue ou un andrologue pour évaluer (i) le volume et la consistance des testicules, (ii)les canaux déférents et les épididymes à la recherche d’une absence totale ou partielle ou des nodules, et (iii) la présence de varicocèle ; (4) d’effectuer 2 spermogrammes, conformément aux recommandations de l’Organisation mondiale de la santé (OMS), si le premier présente au moins une anomalie ; (5) une échographie scrotale systématique, pouvant être complétée par une échographie pelvienne endorectale selon la clinique ; (6) un bilan hormonal comportant au moins une détermination de la testostérone sérique et de FSH ; si la testostérone est basse, la LH permettra de différencier hypogonadisme hypogonatrope (central) et hypogonadisme périphérique ; (7) un caryotype chez les hommes infertiles ayant une concentration de spermatozoïdes ≤ 10,106/ml ; (8) l’évaluation des micro délétions du chromosome Y chez les hommes infertiles ayant une concentration de spermatozoïdes ≤ 1,106/ml ; (9) l’évaluation du gène CFTR en cas de suspicion d’agénésie bilatérale ou unilatérale des canaux déférents et des vésicules séminales. La place de l’analyse de l’intégrité de l’ADN du sperme (SCSA, TUNEL par exemple) dans le bilan de l’homme infertile est encore en cours d’évaluation. Conclusion. — Ces recommandations peuvent être appliquées en pratique clinique en routine chez tout homme infertile.

Resume Depuis la parution de la loi de bioethique de 2004, la preservation de la fertilite fait partie du parcours de soins des patientes devant etre traitees par des chimiotherapies et/ou radiotherapie et/ou chirurgie ovarienne gonadotoxiques et des patientes pour lesquelles la fertilite risque d’etre prematurement alteree. Il existe plusieurs techniques de preservation de la fertilite feminine, dont trois techniques d’assistance medicale a la procreation : la cryoconservation embryonnaire, d’ovocytes isoles et de cortex ovarien. Le choix de la ou des techniques depend du type de traitement, des doses et de sa duree, mais aussi de l’âge de la patiente, de son statut marital, de l’urgence de la mise en place de son traitement anticancereux et de l’hormonosensibilite de la tumeur. Les resultats de ces techniques, dans le cadre de la preservation de la fertilite, sont en pleine evolution et compte tenu d’une utilisation differee des embryons, ovocytes et fragments ovariens cryoconserves, ne sont pas encore definitifs.

International audience

To the editor: The prognosis of sickle cell anemia (SCA), initially disastrous in the severe forms of the disease, has greatly improved, leading to increased life expectancy. More than 95% to 99% of the children diagnosed with SCA in developed countries will become young adults, reaching

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.

PURPOSE: To explore the three-dimensional (3D) organization of sperm genome in DPY19L2-deficient globozoospermic patients speculating a link between DPY19L2 and genome organization of sperm nucleus. METHODS: This is a study of chromatin organization in DPY19L2-deficient globozoospermic patients and healthy donors using three-dimensional fluorescence in situ hybridization (3D-FISH) combined with confocal laser scanning microscopy followed by 3D image analysis. The 3D structures of sperm nuclei, chromocenter, telomeric regions and chromosome territories (CTs), were reconstructed using IMARIS software, and the relative radial position for each individual signal was calculated. Statistical analysis used a non-parametric Mann-Whitney test was appropriate with significance at p

Background: Sperm parameters alteration is documented in untreated men with sickle cell disease (SCD). An aggravating effect of hydroxyurea (HU) on sperm parameters is also well established: HU, at current doses, causes significant, rapid, and unpredictable impairment of spermatogenesis. Reversal of its effect when treatment is stopped has also been documented, albeit in patients given very low doses of treatment (10 mg/kg/d). In France, sperm banking is free of charge and recommended whenever possible before initiation of HU treatment. While recent guidelines have broadened the indication of HU for asymptomatic infantssuch banking is impossible in younger boys before puberty. In addition, little is known on the effect of HU on sperm parameters when given at this specific period. Objectives and Methods: The main objective of this study was to compare sperm parameters after treatment resolution in young males treated by HU prior to puberty with sperm parameters of untreated males. Secondary objective was to analyze longitudinally sperm parameters during HU washout in those treated prior to puberty.Data regarding indication of HU, dosage, date of initiation and stop, clinical profile including vaso occlusive events (VOE) transfusion episodes and date of puberty was collected. Alternative treatment before or during semen analysis was also documented. A period of 3 months of HU wash out was required prior to semen analysis in treated patients. Results: A total of 26 patients (43 semen samples) were studied, with 16 patients treated with HU prior to puberty (HU-PP) and 10 untreated (HU-naive). Characteristics of patients are presented in Table 1. Indication of HU was cerebral vasculopathy (n=3), VOE (n=5), severe anemia (n=1) or combined (n= 7). The median stopping of the HU before CECOS is 4.5 years [0.5-11.0]. An alternative treatment based on a transfusion program was initiated in 14 patients (87.5%) at the time of sperm analysis in the HU-PP group versus 5 patients (50%) in the HU-naive group. Duration of transfusion program was 126 months [3-188] in HU-PP versus 13 [7-100] in the HU naive group. We compared the fraction of abnormal values in semen samples in both groups (25 samples in the HU-PP group and 18 in the HU-naive). No significant difference was observed regarding volume of ejaculate, spermatozoid concentration, total sperm count, spermatozoid motility, morphology and vitality, and sexual abstinence before sampling in both groups (Table 1). In the HU-PP group, there was a trend in improvement of sperm parameters with the duration of transfusion program. In addition, a kinetic analysis of sperm parameters during the period of HU wash out was performed in 3 patients exposed to HU prior to puberty, demonstrating reversibility of HU toxicity in all. All 26 patients were offered semen cryopreservation. For 22 of them, semen parameters after thawing indicated a possible use for assisted reproductive technologies, mostly in vitro fertilization with intracytoplasmic sperm injection. In the remaining 4 (3 in HU-PP group and 1 in HU naïve), there was serious concern about the possible use because of a very low initial sperm concentration and the absence of motile spermatozoids after thawing. Discussion/Conclusion: Toxicity of HU upon spermatogenesis has been well documented in animal studies and in adult males. In many settings, such issues may be a drawback for parents and/or caregivers to treat young boys before semen banking can be performed, given the lack of robust information on reversibility after treatment resolution. Abnormalities of sperm count are also common in non-treated males so that demonstrating the specific effect of HU may be complex. Oseggbe et al. and Berthaud et al. showed that 91% of untreated SCD males have at least one abnormal sperm parameter. Here, we show that after treatment resolution, there are no differences in sperm parameters in patients exposed to HU before puberty compared to untreated males. Because a majority of patients benefitted from transfusion therapy at the time of analysis, we were however unable to demonstrate whether reversal of toxicity occurs spontaneously or requires transfusion. Notwithstanding possible limitations due to small sample size, this study shows that in boys with severe disease requiring HU treatment before puberty, toxicity of HU on sperm parameters should not be a major drawback. Disclosures Bernaudin: AddMedica: Honoraria, Other: Help for travel; GBT: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Consultancy. De Montalembert:Novartis: Consultancy, Honoraria; Addmedica: Consultancy, Honoraria; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Brousse:bluebird bio: Consultancy; Add medica: Consultancy.

The last three authors contributed equally to this work. Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) may improve long-term survival of patients with acute lymphoblastic leukemia (ALL) at high risk of relapse. However, ovarian failure is observed in 70 to 90% of patients who received myeloablative HSCT. Autotransplantation of cryopreserved ovarian cortex harvested before gonadotoxic treatments has been shown to re-establish the menstrual cycles and lead to the birth of healthy children (Donnez J. N Engl J Med, 2018). In patients treated for acute leukemia, concern has been expressed about the risk of leukemia recurrence after the ovarian autotransplantation due to the potential presence of residual leukemic cells in the ovarian tissue (Dolmans MM. Blood, 2010). To date, there were only few studies evaluating the presence of leukemic cells in cryopreserved ovarian tissue, and in most cases ovarian tissues were harvested at diagnosis of ALL or early after onset of chemotherapy. The present study prospectively investigated, in the context of fertility preservation, the presence of leukemic cells in cryopreserved ovarian samples harvested before allogeneic HSCT in patients in complete remission (CR) of ALL. Patients and Methods: From October 2015 to May 2018, all female patients with ALL who had a leukemia specific marker and underwent ovarian cryopreservation before allogeneic HSCT as part of preservation fertility program in 3 centers were included in the study. Consents were obtained from the guardians and/or age appropriate patients. The specific local ethical committees approved the study. Ovariectomy was performed after patients achieved CR, mostly in the weeks preceding HSCT. Ovarian cortex was separated into several fragments and cryopreserved as previously described (Poirot C. Human Reprod, 2002). When more than 14 cortical fragments were obtained, one was dedicated to molecular analysis, as well as the ovarian medulla. MRD quantification was performed by quantitative PCR of clonal rearrangements of immunoglobulin/T-cell receptor genes (Ig/TCR) or oncogenic fusion genes, according to EuroMRD and European Against Cancer (EAC) guidelines. MRD in ovarian samples were compared to MRD in bone marrow (BM) sample obtained at the same time point. Results: Fourteen patients were included in the study: 12 with B-cell precursor ALL, 1 with T-cell ALL and 1 with mixed phenotype acute leukemia. MRD marker was Ig/TCR rearrangement in 12 cases, M-BCR-ABL transcript in 1 case and genomic MLL-AF4 in 1 case. Median age at transplant was 18.2 years (range 1.1 - 35.2 years). Criteria for HSCT were poor early response to treatment (N=8), Philadelphia positive ALL (N=1), or previous relapse (N=5). With a median follow-up after HSCT of 8.1 months (range 0.8 - 29.8 months), all patients are disease-free. At the time of ovariectomy, 7/14 (50%) patients had undetectable MRD in ovarian samples, 6 had low positivity below 10-4 and one had positive MRD higher than 10-4. Unexpectedly of the 7 patients with positive ovarian MRD, 4 were undetectable in BM. One of the 7 patients with undetectable MRD in ovarian samples was positive in bone marrow. Concordant results were observed between the cortex and medulla samples in 10 of the 11 cases where both could be tested. Conclusions: To our knowledge, this series is the largest cohort evaluating MRD in ovarian samples from patients with ALL in complete remission after full chemotherapy regimen. We detected low levels of residual leukemic cells in ovarian tissues in half of the patients, some of them having no detectable MRD in BM at the time of ovariectomy, suggesting that leukemic cells could preferentially persist in ovary. Our results warrant further analyses of an extended cohort and longer post-transplant follow-up to better assess the potential presence of leukemic cells in ovarian samples and evaluate the impact of ovarian MRD status on post HSCT relapse. Still, autotransplantation of cryopreserved ovarian cortex could be discussed after allogeneic HSCT in patients with undetectable ovarian MRD. Table 1. MRD evaluation in bone marrow and ovarian samples. BM, bone marrow; BCP-ALL, B-cell precursor ALL; Ph-positive ALL, ALL with Philadelphia chromosome; NA: not available. Undetectable MRD means negative result obtained with a sensitivity of 10-4 or 10-5. BCR-ABL1 MRD results are expressed as the BCR-ABL1/ABL1 transcripts ratio. Disclosures No relevant conflicts of interest to declare.

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin alpha6beta1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of alphavbeta3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-alphav or anti-beta3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm alphavbeta3 integrin.

Background: Genomic imprinting refers to an epigenetic marking resulting in monoallelic gene expression and has a critical role in fetal development. Various imprinting diseases have recently been reported in humans and animals born after the use of assisted reproductive technology (ART). All the epimutations implicated involve a loss of methylation of the maternal allele (demethylation of KvDMR1/ KCNQ1OT1 in Beckwith–Wiedemann syndrome (BWS), demethylation of SNRPN in Angelman syndrome and demethylation of DMR2/ IGF2R in large offspring syndrome), suggesting that ART impairs the acquisition or maintenance of methylation marks on maternal imprinted genes. However, it is unknown whether this epigenetic imprinting error is random or restricted to a specific imprinted domain. Aim: To analyse the methylation status of various imprinted genes ( IGF2R gene at 6q26, PEG1/MEST at 7q32, KCNQ1OT1 and H19 at 11p15.5, and SNRPN at 15q11–13) in 40 patients with BWS showing a loss of methylation at KCNQ1OT1 (11 patients with BWS born after the use of ART and 29 patients with BWS conceived naturally). Results: 3 of the 11 (27%) patients conceived using ART and 7 of the 29 (24%) patients conceived normally displayed an abnormal methylation at a locus other than KCNQ1OT1 . Conclusions: Some patients with BWS show abnormal methylation at loci other than the 11p15 region, and the involvement of other loci is not restricted to patients with BWS born after ART was used. Moreover, the mosaic distribution of epimutations suggests that imprinting is lost after fertilisation owing to a failure to maintain methylation marks during pre-implantation development.

La prise en charge de l’infertilite a debute il y a plus de deux siecles par la realisation d’inseminations artificielles, technique interferant peu sur le processus de reproduction puisque la fecondation se deroulein vivo. Les annees 80 ont vu l’emergence de la fecondationin vitro (FIV) necessitant une stimulation hormonale soutenue, une fecondation exvivo et une culture embryonnaire. Enfin, l’arrivee de l’ICSI (Intra Cytoplasmic Sperm Injection) a revolutionne la prise en charge des couples en permettant de creer un embryon par simple microinjection d’un spermatozoide, meme anormal ou immature, dans un ovocyte. Rapidement, la question de l’impact de ces techniques sur la sante des enfants ainsi concus a ete posee. Des etudes recentes ont suggere un lien entre aide medicale a la procreation et developpement de pathologies d’empreinte. En effet, l’empreinte parentale etablie specifiquement sur chacun des gametes et transmise a l’embryon peut etre sensible a des facteurs externes. La manipulation et la culturein vitro des gametes et embryons, inherentes a la realisation de la FIV/ICSI, pourraient avoir des consequences sur l’etablissement et/ou la conservation de l’empreinte. La connaissance des mecanismes d’acquisition et de maintien de l’empreinte et l’analyse de leur deregulation sont necessaires pour evaluer ce risque potentiel.

Significant production of the growth factor IGF2 has been reported in human hepatocellular carcinomas (HCCs). Disturbances associated with changes in methylation at this locus or affecting the 11p15.5 imprinting domain as a whole can be postulated in HCCs. In the present study, the methylation status of differentially methylated regions of the imprinted genes TSSC5, LIT1, and IGF2, which span the 11p15 domain, was analysed in 71 liver tissues from virus-associated and non-virus-associated HCCs compared with six normal liver tissues. Altered methylation of TSSC5 and LIT1 was observed in only 6% and 8% of HCCs, respectively, compared with 89% at the IGF2 locus, suggesting that these loci were not concomitantly dysregulated. These observations suggest that loss of parental-specific methylation at the IGF2 locus may be specifically associated with HCC, whether virus-associated or non-virus-associated, and arising in cirrhotic or non-cirrhotic livers. Copyright © 2003 John Wiley & Sons, Ltd.

Objective: To provide a link between epigenetics and male subfertility at the DNA, histone-protamine, and RNA levels and its consequences on fertilization and embryo development. Design: Review of the relevant literature. Setting: University-based clinical and research laboratories. Patient(s): Fertile and infertile men. Intervention(s): None. Main Outcome Measure(s): Critical review of the literature. Result(s): Epigenetic markers can be modified in infertile patients. Epigenetic modifications include methylation loss or gain on the global level and on imprinted genes, high levels of histone retention in spermatozoa, and deficiencies in some transcripts involved in spermatogenesis. Interestingly, these abnormalities are all linked together, because DNA methylation maintenance depends on DNA histone-protamine configuration which itself is stabilized by spermatozoal RNAs. Conclusion(s): The paternal genome has long been considered to be silent and passive in embryo formation. The epigenetic processes associated with the paternal DNA genome highlights its importance in male fertility as well as for embryo development.

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.

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.

OBJECTIVE: To review donor-egg assisted reproductive technology (ART) activity using young fertile donors (

Following the death in France by acute aortic dissection of two women with Turner syndrome who were pregnant following oocyte donation, the Director of the French Biomedicine Agency (Agence de la biomedecine) sent a letter to the President of the French College of Obstetricians and Gynaecologists (FCOG). He requested the College's expertise in reviewing point-by-point the cases and risk factors and in determining whether there are grounds to propose additional measures complementary to the recommendations made by the Haute autorite de sante or French National Authority for Health (HAS) in 2008 in terms of indication and monitoring of patients. A joint practice committee of the FCOG, the French Cardiologic Society, the French Chest and Cardiovascular Surgery Society, the French Society of Anaesthesia and Intensive Care, the French Endocrine Society, the French study group for oocyte donation, and the Biomedicine Agency defined the exact questions to be put to the experts, chose these experts, followed them up and drafted the synthesis of recommendations resulting from their work. The questions concerned the check-up before pregnancy of Turner patients, contraindication and acceptance of pregnancy, information for the patients, and recommendations for antenatal care, delivery and postnatal follow-up.

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin alpha6beta1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of alphavbeta3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-alphav or anti-beta3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm alphavbeta3 integrin.

The objective of this retrospective study was to describe a population of patients displaying impaired sperm motility due to ultrastructural flagellar defects and to analyse the intracytoplasmic sperm injection (ICSI) results and neonatal outcomes in this population. The fertilization rate, embryo quality, clinical pregnancy rate, implantation rate, birth rate and perinatal health of babies were determined. Patients (n = 20) were divided into seven categories according to ultrastructural flagellar abnormalities. The type of flagellar abnormality significantly affected the fertilization rate (P

Medical taking part in procreation in gamete and embryo donation is recent in France and has been included in the field of medically assisted procreation as defined by a law since 1994. Theses activities are strictly regulated. If procreation using donor sperm has been broadly carried out for 30 years in the CECOS leading to the birth of approximately 40,000 children, oocyte and embryo donation had a more recent and limited development. The main questions to which are faced the couples procreating through gamete and embryo donation is whether their child should be told about its origin since they have to build a parenthood and a filiation which are not first depending on biological and genetic links.

Significant production of the growth factor IGF2 has been reported in human hepatocellular carcinomas (HCCs). Disturbances associated with changes in methylation at this locus or affecting the 11p15.5 imprinting domain as a whole can be postulated in HCCs. In the present study, the methylation status of differentially methylated regions of the imprinted genes TSSC5, LIT1, and IGF2, which span the 11p15 domain, was analysed in 71 liver tissues from virus-associated and non-virus-associated HCCs compared with six normal liver tissues. Altered methylation of TSSC5 and LIT1 was observed in only 6% and 8% of HCCs, respectively, compared with 89% at the IGF2 locus, suggesting that these loci were not concomitantly dysregulated. These observations suggest that loss of parental-specific methylation at the IGF2 locus may be specifically associated with HCC, whether virus-associated or non-virus-associated, and arising in cirrhotic or non-cirrhotic livers.

International audience; In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of “fingerprint,” revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

Abstract 242 Introduction Hydroxyurea (HU) has proved its efficacy in reducing vaso-occlusive events in patients with SCD and increasing life expectancy. However, effects on fertility in adult males represent a major issue for acceptance and adherence, as HU has been reported to impair spermatogenesis through direct cytotoxic effects and hypogonadism. There are only limited data in the literature in this field and mainly retrospective. Patients and methods 49 adult patients aged 20–52 years with homozygous SCD eligible for first line HU treatment were enrolled after informed consent between June 2010 and April 2012 in this prospective study: HYDREP.The main objective was to compare at Day 0 and 180 of HU treatment the semen parameters according to the WHO (1999) criteria(volume, sperm concentration, viability, forward motility and morphology ). Vaso-occlusive events and other complications, number of transfusions during the 6 months preceding and following HU initiation were recorded. Hematological, biochemical, hormonal parameters, HU dosage were recorded at Day 0, Day 90 and 180. HU was begun at 15mg/Kg dose (Platt *) and modulated following haematological tolerance and renal function. Results and discussion Preliminary results concern the 24/49 patients, who complete the 6 months protocol. A statistically significant impairment in all sperm parameters was observed, after a 6 months HU therapy, with great variations between individuals, which are to be explained by complementary tests. These preliminary deleterious findings must be interpreted with caution as in our experience 1) spermatogenesis may recover after drug withdrawal 2) Repeated vaso-occlusive events are also potentially deleterious on fertility (Berthaut **)and 3)this study must be completed with a survey on effective fertility seen in men treated with HU. Thus, regarding to the great benefit offered by HU on prognosis allowing a real “life project” in young symptomatic adults with SCD, it is worth providing each patient of the most accurate information on fertility as an essential prerequisite for their adhesion to HU treatment. Conclusion Sperm cryopreservation is advised before beginning a treatment with HU. Prospective follow up studies of HU male cohort regarding fertility are warranted. Health education has a major role to play to increase HU compliance especially in view of these results. Disclosures: No relevant conflicts of interest to declare.