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Non-obstructive azoospermia (NOA) is a severe and frequent cause of male infertility, often treated by testicular sperm extraction followed by intracytoplasmic sperm injection. The aim of this study is to improve the genetic diagnosis of NOA, by identifying new genes involved in human NOA and to better assess the chances of successful sperm extraction according to the individual’s genotype. Exome sequencing was performed on 96 NOA-affected individuals negative for routine genetic tests. Bioinformatics analysis was limited to a panel of 151 genes selected as known causal or candidate genes for NOA. Only highly deleterious homozygous or hemizygous variants were retained as candidates. A likely causal defect was identified in 16 genes in a total of 22 individuals (23%). Six genes had not been described in man (DDX25, HENMT1, MCMDC2, MSH5, REC8, TDRKH) and 10 were previously reported (C14orf39, DMC1, FANCM, GCNA, HFM1, MCM8, MEIOB, PDHA2, TDRD9, TERB1). Seven individuals had defects in genes from piwi or DNA repair pathways, three in genes involved in post-meiotic maturation, and 12 in meiotic processes. Interestingly, all individuals with defects in meiotic genes had an unsuccessful sperm retrieval, indicating that genetic diagnosis prior to TESE could help identify individuals with low or null chances of successful sperm retrieval and thus avoid unsuccessful surgeries.

BACKGROUND Many studies reported that reproductive desire could be high among transgender individuals. In France, fertility preservation and sperm donation were very little proposed to transgender individuals until recently, mainly because the Bioethics Law allows the use of assisted reproductive technologies only in infertile couples and prohibits surrogacy. OBJECTIVES To evaluate the distribution of care on the French territory concerning fertility preservation and sperm donation in transgender individuals. MATERIALS AND METHODS A multicentric national survey was carried out between January 2019 and October 2020 in 28 assisted reproductive technology centres of the French CECOS (Centres d'Etudes et de Conservation des Oeufs et du Sperme) network. Each centre was questioned to find out how many transgender individuals came, were informed and cared for fertility preservation and sperm donation. RESULTS Concerning fertility preservation, 71.4% of centres received transgender individuals and performed gamete cryopreservation; 581 transgender individuals consulted for fertility preservation. Transgender women were more likely to desire (p

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.

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.

International audience; Globozoospermia is a rare phenotype of primary male infertility inducing the production of round-headed spermatozoa without acrosome. Anomalies of DPY19L2 account for 50-70% of all cases and the entire deletion of the gene is by far the most frequent defect identified. Here, we present a large cohort of 69 patients with 20-100% of globozoospermia. Genetic analyses including multiplex ligation-dependent probe amplification, Sanger sequencing and whole-exome sequencing identified 25 subjects with a homozygous DPY19L2 deletion (36%) and 14 carrying other DPY19L2 defects (20%). Overall, 11 deleterious single-nucleotide variants were identified including eight novel and three already published mutations. Patients with a higher rate of round-headed spermatozoa were more often diagnosed and had a higher proportion of loss of function anomalies, highlighting a good genotype phenotype correlation. No gene defects were identified in patients carrying < 50% of globozoospermia while diagnosis efficiency rose to 77% for patients with > 50% of globozoospermia. In addition, results from whole-exome sequencing were scrutinized for 23 patients with a DPY19L2 negative diagnosis, searching for deleterious variants in the nine other genes described to be associated with globozoospermia in human (C2CD6, C7orf61, CCDC62, CCIN, DNAH17, GGN, PICK1, SPATA16, and ZPBP1). Only one homozygous novel truncating variant was identified in the GGN gene in one patient, confirming the association of GGN with globozoospermia. In view of these results, we propose a novel diagnostic strategy focusing on patients with at least 50% of globozoospermia and based on a classical qualitative PCR to detect DPY19L2 homozygous deletions. In the absence of the latter, we recommend to perform whole-exome sequencing to search for defects in DPY19L2 as well as in the other previously described candidate genes.

Context Prokineticin 1 (PROK1) quantification in global follicular fluid (FF) has been recently reported as a predictive biomarker of in vitro fertilization (IVF) outcome. It is now necessary to evaluate its clinical usefulness in individual follicles. Objectives To evaluate the clinical value of PROK1 secretion in individual FF to predict oocyte competence. To determine the impact of follicular size, oocyte maturity, and gonadotropin treatments on PROK1 secretion. Design and setting Prospective cohort study from May 2015 to May 2017 at the University Hospital of Grenoble. Patients A total of 69 infertile couples underwent IVF. Intervention(s) Collection of 298 individual FF from 44 women undergoing IVF; 52 individual cumulus cell (CC) samples and 15 CC primary cultures from 25 women undergoing IVF-intracytoplasmic sperm injection (ICSI). Main Outcome Measure(s) Oocyte competence was defined as the ability to sustain embryo development to the blastocyst stage. Follicular size was measured by 2D-sonography. PROK1 concentration was quantified by ELISA assay. Results PROK1 concentration was correlated to follicular size (r = 0.85, P = 2.2 × 10−16). Normalized PROK1 concentration in FF was predictive of subsequent oocyte competence (AUROC curve = 0.76 [95% CI, 0.69–0.83]; P = 1.7 × 10−9), irrespectively of day-2 embryo morphokinetic parameters. The expression and secretion of PROK1 were increased in FF and CC of mature oocytes (P < 0.01). Follicle Stimulating Hormone and hCG up-regulated PROK1 secretion in CC primary cultures (P < 0.01; P < 0.05), probably through the cAMP pathway (P < 0.01). Conclusions PROK1 quantification in individual FF could constitute a new predictive biomarker of oocyte competence in addition with embryo morphokinetic parameters. Trial registration number none.

Background Sterility is a major late effect of radiotherapy and chemotherapy treatments. Iatrogenic sterility is often permanent and greatly impacts long-term quality of life. Ovarian tissue cryopreservation (OTC) performed before gonadotoxic treatments with subsequent autograft is a method of fertility preservation available for girls and women. Its application in prepubertal girls is of particular value as it is the only possible approach in this patient group. In addition, it does not require a delay in cancer therapy and no ovarian stimulation is needed. Objective The primary aim of this protocol is to help increase the implementation of ovarian tissue autografting in France. Knowledge is still lacking regarding the efficacy of ovarian transplantation in restoring ovarian function and regarding the safety of this procedure, especially the risk of cancer cell reseeding in certain types of cancer. A secondary aim of this study is to generate data to improve our understanding of these two essential aspects. Methods The DATOR (Development of Ovarian Tissue Autograft in Order to Restore Ovarian Function) study is ongoing in 17 university hospitals. The DATOR protocol includes the autograft of ovarian cortex fragments. Candidates are identified from an observational prospective cohort (called the Prospective Cohort of Patients Candidates for Ovarian Tissue Autograft [PERIDATOR]) of patients who have undergone OTC. Enrollment in the study is initiated at the patient’s request and must be validated by the center’s multidisciplinary team and by the study steering committee. The DATOR study begins with a total medical checkup. Ovarian tissue qualification and residual disease detection, if required, are performed. Results The study is ongoing. Currently, 38 patients have provided informed consent and have been entered into the DATOR study. Graft has been performed for 34 of these patients. An interim analysis was conducted on the first 25 patients for whom the period of at least 1 year posttransplantation was achieved. Out of these 25 patients, 11 women succeeded in becoming pregnant (pregnancy rate=44% [11/25]; delivery rate=40% [10/25]). Among these, 6 women conceived twice, and 1 pregnancy led to a miscarriage. Conclusions Our preliminary analysis appears to be coherent with the accumulating body of evidence indicating the potential utility of ovarian tissue autograft for patients with premature ovarian failure. All these elements justify the pursuit of our study. Trial Registration ClinicalTrials.gov NCT02846064; https://clinicaltrials.gov/ct2/show/NCT02846064 International Registered Report Identifier (IRRID) DERR1-10.2196/12944

International audience; Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.

STUDY QUESTION Does DNAH1 status influence intracytoplasmic sperm injection (ICSI) outcomes for patients with multiple morphological abnormalities of the sperm flagella (MMAF)? SUMMARY ANSWER Despite a highly abnormal morphology, sperm from MMAF patients with DNAH1 mutations have a low aneuploidy rate and good nuclear quality, leading to good embryonic development following ICSI and a high pregnancy rate. WHAT IS KNOWN ALREADY Teratozoospermia represents a heterogeneous group including a wide range of phenotypes. Among all these qualitative defects, a flagellar phenotype called MMAF is characterized by a mosaic of morphological abnormalities of the flagellum, including coiled, bent, irregular, short or/and absent flagella, mainly due to the absence of the axonemal central pair microtubules. We previously demonstrated that homozygous mutations in the DNAH1 gene, encoding an inner arm heavy chain dynein, are frequently found in patients with MMAF (28% of the patients from the initial cohort). Numerous studies have reported an increased rate of aneuploidy and a poor sperm nuclear quality related to sperm flagellar abnormalities, which could impede ICSI outcome. Moreover, success rates after ICSI may be influenced by the type of ultrastructural flagellar defects and/or by the gene defects carried by the patients. STUDY DESIGN, SIZE, DURATION This retrospective cohort study included 6 infertile males with MMAF due to deleterious homozygous DNAH1 mutations and their respective spouses, who underwent 9 ISCI cycles, with 16 embryos being transferred. ICSI results were compared with two control populations of 13 MMAF men without DNAH1 mutations and an aged-matched control group of 1431 non-MMAF couples. All ICSI attempts took place between 2000 and 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS Clinical and biological data were collected from patients treated for infertility at the CPSR les Jasmins in Tunis (Tunisia). We compared the ICSI outcomes obtained with couples including DNAH1 mutated and nonmutated patients and non-MMAF couples. For the analysis of the chromosomal status, fluorescence in situ hybridization (FISH) analyses were performed on sperm cells from 3 DNAH1-mutated patients and from 29 fertile control subjects. Sperm chromatin condensation and DNA fragmentation were evaluated using aniline blue staining and TUNEL assays, respectively, on sperm cells from 3 DNAH1-mutated men and 6 fertile controls. MAIN RESULTS AND THE ROLE OF CHANCE There was a significantly increased proportion of disomy XY and 18 in sperm from DNAH1 mutated patients compared with fertile controls (1.52 versus 0.28%, P = 0.0001 and 0.64 versus 0.09%, P = 0.0001). However, there were no statistically significant differences among sperm from the two groups in their frequencies of either 13, 21, XX or YY disomy or diploidy. Measures of DNA compaction and fragmentation demonstrated a good nuclear sperm quality among DNAH1 mutated men. The overall fertilization, pregnancy and delivery rates of couples including DNAH1 mutated men were of 70.8, 50.0 and 37.5%, respectively. There were no statistically significant differences in any of these parameters compared with the two control groups (P > 0.05). LIMITATIONS, REASONS FOR CAUTION A limitation of this study is the small number of DNAH1-mutated patients available and the low number of genes identified in MMAF. Further genetic studies are warranted to identify other MMAF-inducing genes to better characterize the genetic etiology of the MMAF phenotype and to improve the management of patients diagnosed with flagellar defects. WIDER IMPLICATIONS OF THE FINDINGS MMAF patients with DNAH1 mutations have low aneuploidy rates and good nuclear sperm quality, explaining the high pregnancy rate obtained with these patients. Good ICSI results were obtained for both MMAF groups (DNAH1 mutated and nonmutated), suggesting that patients presenting with asthenozoospermia due to flagellar defects have a good ICSI prognosis irrespective of their genotype. The majority of MMAF cases currently remain idiopathic with no genetic cause yet identified. In depth genetic analysis of these patients using next generation sequencing should reveal new causal genes. Subsequent genotype phenotype analyses could improve advice and care provided to MMAF patients. STUDY FUNDING/COMPETING INTERESTS None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)', funded by the program GENOPAT 2009 from the French Research Agency (ANR) and the MAS-Flagella project, financed by the French ANR and the Direction Generale de l'Offre de Soins (DGOS).

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2β and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2β is critical for spontaneous AR, whereas both iPLA2β and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0–5 min post-P4) rely on iPLA2β, whereas sperm undergoing late AR (20–30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (∼10 μm).

The purpose of the present multicenter study was to investigate whether an artificial insemination with donor sperm (AID) procedure after intra-couple intracytoplasmic sperm injection (ICSI) failure offers a significant chance of pregnancy and to identify prognostic factors for pregnancy after an AID procedure. An eleven-year retrospective multicenter study was conducted among 13 Centre d'Etude et de Conservation des Oeufs et du Sperme (CECOS) centers. A total of 319 couples having undergone an AID procedure after intra-conjugal ICSI failure were included in this study; a total of 1,159 AID and 1,011 intra-conjugal ICSI cycles were performed. Among the prognostics parameters, the parity and the embryo quality could not be adequately addressed, therefore the parity was not included in the statistical analysis and the embryo quality has been presented as preliminary observations. The pregnancy rate per cycle was 12.0% (139/1,159) and the overall AID pregnancy rate per couple was 43.6% (139/319). Normal or oligoasthenoteratozoospermia (OAT) semen and women aged 34 years or above at the time of AID procedure obtained the lowest AID clinical pregnancy rate. Azoospermia or cryptozoospermia semen and women aged below 34 years obtained the highest AID clinical pregnancy rate. In conclusion, the transition to the AID procedure after intra-conjugal ICSI failure allows such couples to obtain a pregnancy, however after each ART failure AID transition should be proposed according to the woman's age and sperm characteristics.AID: artificial insemination with donor sperm; ICSI: intracytoplasmic sperm injection; CECOS: Centre d'Etude et de Conservation des Oeufs et du Sperme; OAT: oligoasthenoteratozoospermia; IVF: in vitro fertilization; ART: artificial reproductive technology; β hCG: beta human chorionic gonadotrophin; SD: standard deviation; OR: Odds ratio.

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human., Asthenozoospermia is a major cause of male infertility, and multiple morphological abnormalities of the flagella (MMAF) is a particularly severe form. Here, using whole-exome sequencing of 78 MMAF patients, the authors identify mutations in two WDR proteins, CFAP43 and CFAP44, and confirm that these proteins are required for flagellogenesis in mouse and Trypanosoma brucei.

International audience; Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes.

International audience; OBJECTIVE: To assess sperm production and aneuploidy in Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) before and after treatments. DESIGN: Multicenter, prospective, longitudinal study of lymphoma patients analyzed before treatment and after 3, 6, 12, and 24 months. SETTING: University hospitals. PATIENT(S): Forty-five HL and 13 NHL patients were investigated before and after treatment. Treatment regimens were classified in two groups: ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) with or without (+/-) radiotherapy, and CHOP (doxorubicin, cyclophosphamide, vincristine, prednisone)/MOPP-ABV (mechlorethamine, oncovin, procarbazine, prednisone-doxorubicin, bleomycin, vinblastine). A control group of 29 healthy men was also studied. INTERVENTION(S): Semen analyses and aneuploidy study by FISH were performed at each time point. MAIN OUTCOME MEASURE(S): Comparison of mean sperm characteristics and percentage of sperm aneuploidy rates before and after treatment. RESULT(S): Before treatment, HL and NHL men had altered semen characteristics and higher sperm aneuploidy rates (median 0.76 [interquartile range 0.56-0.64]) than the control group (0.54 [0.46-0.74]). After treatment, sperm production was significantly lowered 3 and 6 months after ABVD +/- radiotherapy or CHOP/MOPP-ABV. After ABVD +/- radiotherapy, the aneuploidy rate increased significantly only at 3 months, and values obtained 1 or 2 years later were lower than pretreatment values. In contrast, in the CHOP/MOPP-ABV treatment group, semen characteristics and aneuploidy rate did not return to normal levels until 2 years after treatment. CONCLUSION(S): Lymphoma itself has consequences on sperm aneuploidy frequency before treatment. Moreover, lymphoma treatments have deleterious effects on sperm chromosomes related to treatment type and time since treatment. Patient counseling is essential concerning the transient but significant sperm aneuploidy induced by lymphoma and its treatments.

International audience; The aim of this study was to characterize the nuclear lamina (NL) and lamin chromatin-partners in spermatozoa from four DPY19L2-deleted globozoospermic patients. We tested for spermatid transcripts encoding lamins and their chromatin-partners emerin, LAP2a, BAF and BAF-L, by reverse transcriptasePCR using spermatozoa RNA. We also determined the localization of lamin B1, BAF and BAF-L by immunofluorescent analysis of spermatozoa from all patients. In RNA from globozoospermic and control spermatozoa we detected transcripts encoding lamin B1, lamin B3, emerin, LAP2a and BAF-L, but not A-type lamins. In contrast, BAF transcripts were detected in globozoospermic but not control spermatozoa. The NL was immature in human globozoospermic spermatozoa: lamin B1 signal was detected in the nuclei of globozoospermic spermatozoa in significantly higher proportions than the control (P < 0.05; 56-91% versus 40%) and was predominantly observed at the whole nuclear periphery, not polarized as in control spermatozoa. Conversely, BAF and BAF-L were detected in control, but not globozoospermic spermatozoa. Our results strongly emphasize the importance of the NL and associated proteins during human spermiogenesis. In globozoospermia, the lack of maturation of the NL, and the modifications in expression and location of chromatin-partners, could explain the chromatin defects observed in this rare phenotype. (C) 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospho- lipase C, zeta (PLCz), the sperm factor thought to induce the Ca 2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCz in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCz and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCz is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca 2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCz in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equa- torial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCz in globozoospermic sperm is thus con- sistent and reinforces the role of PLCz as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

Resume Objectifs Evaluer differentes techniques chirurgicales d’ovariectomie partielle pour cryopreservation. Evaluer les consequences d’une exposition prealable aux traitements cytotoxiques sur la qualite de l’ovaire preleve. Patientes et methodes Etude observationnelle retrospective unicentrique, sur 4 ans, des femmes ayant eu une chirurgie pour cryopreservation ovarienne pour chimio ou radiotherapie a haut risque de faillite ovarienne precoce. Plusieurs techniques d’ovariectomie partielle ont ete proposees : avec clampage du pedicule vasculaire gonadique (prelevement indirect), sans clampage (prelevement direct) et a la pince agrafeuse automatique. Le tissu ovarien etait immediatement prepare pour la cryoconservation en salle d’operation. L’ensemble du prelevement etait divise en petits fragments. Pour chaque ovaire, un compte des fragments etait effectue. Un fragment etait examine afin de determiner le nombre de follicules primordiaux. Resultats L’ovariectomie et la cryopreservation ont ete realisees chez 13 patientes. Deux evenements hemorragiques sont survenus avec la technique directe, sans consequence pour les patientes. Le nombre de fragments obtenus avec les techniques indirecte et directe etait de respectivement 19 vs 15, p = 0,18 ; le nombre de follicules primordiaux etait de 38 vs 36, p = 0,87. L’agrafeuse automatique consommait trop de tissu ovarien pour etre interessante. Il y avait moins de fragments, 15 vs 20, p Discussion et conclusion La technique avec clampage vasculaire est plus sure mais sans difference sur la qualite du prelevement. Une seule cure de chimiotherapie a un impact pejoratif sur la qualite du prelevement.

In spite of improvements in assisted reproductive technology (ART) during the last 30 years, the rate of pregnancy remains constrained, as only about 25 % of embryo transfer lead to successful pregnancies, even with an average of two embryos replaced. Embryo selection is currently based on the establishment of morphokinetic scores, a method that obviously exhibits limitations. Therefore, the assessment of embryo development potency by criteria of higher predictive value is mandatory in order to increase the rates of pregnancy. Nowadays, there is increasing evidence that angiogenic factors might contribute to the success of the implantation and to the pregnancy outcome. Among these factors, prokineticin 1 (PROK1) and its receptors (PROKRs) constitute new targets that showed over the last ten years strong biological features directly linked to ovarian physiology, endometrial receptivity, embryo implantation and thus successful pregnancies. In ART, the rates of circulating PROK1 were reported in 2012 as significantly linked to the quality of embryonic cohort, as well as to the rates of pregnancy. Our preliminary data suggest a high potential of this cytokine in the success of implantation and pregnancy, and strongly overtones the emergency to investigate the value of its measurement in conditioned media of oocytes and embryo cultures in ART.

STUDY QUESTION Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus? SUMMARY ANSWER We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments. WHAT IS KNOWN ALREADY Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore, been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced. STUDY DESIGN, SIZE, DURATION This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species. PARTICIPANTS/MATERIALS, SETTING, METHODS For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide. MAIN RESULTS AND THE ROLE OF CHANCE Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called ‘spermaurin’. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide. LIMITATIONS, REASONS FOR CAUTION This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs. STUDY FUNDING/COMPETING INTEREST(S) This work is part of the project ‘LAB COM-14 LAB7 0004 01-LIPAV’, funded by the program LabCom 2014 from t e French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript.

Prokineticin 1 (PROK1), also called endocrine gland-derived vascular endothelial growth factor, is a well-established regulator of endometrial receptivity and placental development. However, its clinical usefulness as a noninvasive predictive biomarker of embryo implantation is yet to be validated.The main objective of this article was to determine the relationship between PROK1 levels in the follicular fluid (FF) and fertilization culture media (FCM) and the reproductive outcome in patients who received a first conventional in vitro fertilization-embryo transfer. The secondary objective was to characterize the expression of PROK1 and its receptors (PROKRs) in the human follicular microenvironment.We conducted a prospective study between January 2013 and June 2015 at the University Hospital of Grenoble.A total of 135 infertile in vitro fertilization patients and 10 women undergoing ovarian tissue cryopreservation were included.The PROK1 concentration was measured by ELISA in FF and FCM collected on the day of oocyte retrieval and the day of the oocyte denudation step, respectively. Follicular expression of the PROK1/PROKR system was determined by immunohistochemistry, RT-quantitative PCR, and ELISA.Assessment of the clinical pregnancy rates was the main outcome.FF and FCM PROK1 levels were significantly higher in the embryo implantation group (P.001) and were predictive of subsequent embryo implantation (area under the receiver operating characteristic curve, 0.91 [95% confidence interval, 0.81-1.00], P = .001; and 0.88 [0.72-1.00], P = .001, respectively). FF and FCM PROK1 levels remain similar irrespective of the embryo morphokinetic parameters (P = .71 and P = .83, respectively). The PROK1/PROKR system is expressed during human folliculogenesis.PROK1 levels in FF and FCM could constitute new predictive noninvasive markers of successful embryo implantation in conventional in vitro fertilization-embryo transfer.

Resume Introduction Cette revue concerne les regles de fonctionnement des CECOS en France et les problemes legaux, medicaux et ethiques souleves par le don de spermatozoides. Materiel et methodes Selection des articles et des conferences de consensus publies sur le theme dans Medline (PubMed) de 1973 a 2011 et retenus en fonction de leur pertinence ainsi que les textes legaux releves sur les sites officiels legislatifs francais. Resultats Les regles de fonctionnement des CECOS ont ete etablies par la loi du 29 juillet 2004, revisee le 6 aout 2004, puis le 7 juillet 2011. Parmi les 21 759 enfants nes d’une AMP en France en 2009, 5,1 % sont issus d’un don de spermatozoides. De 1973 a 2006, 44 045 enfants sont nes apres un don de spermatozoides. Entre 1973 et 2006, 16 971 donneurs se sont presentes dans les CECOS et seulement 10 347 ont effectue completement leur demarche de don. La principale indication de recours au don de spermatozoides (75 % des demandes) est representee par les hommes de couples infertiles avec une azoospermie non obstructive, la seconde indication correspond aux hommes infertiles presentant une oligozoospermie. En cas d’azoospermie, la demande est effectuee le plus souvent apres echec d’un prelevement chirurgical testiculaire ou epididymaire. En cas d’oligozoospermie, la demande intervient le plus souvent apres plusieurs echecs d’AMP intraconjugales. Conclusion De nombreuses interrogations sont encore presentes autour de la conception d’enfants par don de spermatozoides. Celle de la legitimite du maintien de l’anonymat dans le don reste largement debattue.

Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.

International audience; STUDY QUESTION: Can we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation? SUMMARY ANSWER: We identified a new non-sense mutation, p.Y248*, that represents 13% of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC. WHAT IS KNOWN ALREADY: The absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC). STUDY DESIGN, SIZE, DURATION: This is a retrospective study carried out on patients consulting for infertility and described as having >5% large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients consulted for primary infertility in fertility clinics in France (n = 44), Tunisia (n = 30), Morocco (n = 9) or Algeria (n = 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248*. Microsatellite analysis was performed on all p.Y248* patients to evaluate the age of this new mutation. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a new non-sense mutation, p.Y248*, in 10 unrelated individuals of European (n = 4) and North African origin (n = 6). We show that this new variant represents 13% of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5%). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3' untranslated region was found to strictly co-segregate with p.Y248*, demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n = 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Despite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the programme GENOPAT 2009 from the French Research Agency (ANR).

STUDY QUESTION: Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia? SUMMARY ANSWER: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. WHAT IS KNOWN ALREADY: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. STUDY DESIGN, SIZE, DURATION: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. MAIN RESULTS AND THE ROLE OF CHANCE: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. LIMITATIONS, REASONS FOR CAUTION: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. WIDER IMPLICATIONS OF THE FINDINGS: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR).

International audience; An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

Infertility concerns at least 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. Hundreds of genes are probably involved in spermatogenesis and oogenesis and this genetic heterogeneity has so far hindered the identification of genes causing infertility in the human. Careful morphological examination of spermatozoa can provide cues to identify homogeneous cohorts of patients likely to have the same genetic defect. We studied a cohort of North-Africans patients with a rare phenotype of large-headed spermatozoa. Using a homozygosity mapping strategy, we could map the morbid gene and we identified the same homozygous mutation (c.144delC) in the aurora kinase C gene (AURKC) of all patients studied initially. We then genotyped a total of 62 patients. All who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n=34), whereas no AURKC mutations were detected in the others. A carrier frequency of 1/50 was established from individuals from the Maghrebian population, indicating that 1 in 10,000 men from North-African can be expected to present this form of infertility, a frequency comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. Then we demonstrated by flow cytometry that all spermatozoa have in fact a homogeneous 4C. We recommend the realisation of a molecular diagnosis to all patients with large-headed spermatozoa. ICSI is formally contraindicated for all homozygous patients who can have recourse to donor sperm or adoption. One cannot be as categorical for the patients not harbouring an AURKC mutation.

The in vitroO-glycosylation reaction of the MUCSAC mucin motif peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUCSAC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUCSAC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitroO-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2. © Munksgaard 1998.

Le syndrome de Klinefelter est l’une des principales causes genetiques d’infertilite masculine. Il est en effet diagnostique chez 11% des patients presentant une azoospermie et 4% des hommes infertiles [23]. Notre etude rapporte une naissance apres ICSI utilisant du sperme ejacule chez un jeune homme de 21 ans, presentant un syndrome de Klinefelter pur, homogene, decouvert lors d’un bilan d’infertilite pour oligozoospermie severe. Sur une periode de 18 mois, trois ICSI ont ete realisees pour ce couple. Au cours des deux premieres tentatives avec du sperme ejacule frais, 10 embryons au total ont ete transferes sans obtenir de grossesse. Lors de la 3e ICSI, nous avons du utiliser des spermatozoides ejacules congeles, car le patient presentait une azoospermie averee le jour de l’ICSI. Une grossesse a ete obtenue apres transfert de 3 embryons de grade A, avec naissance a ternie depasse d’une fille en bonne sante. Nous soulignons l’interet d’une congelation preventive repetee lorsque des spermatozoides ejacules sont disponibles dans tous les cas d’oligozoospermie severe ou de cryptozoospermie. De plus, nous avons evalue la qualite des spermatozoides congeles et utilisables en ICSI par une analyse de leur ADN et une analyse de leur ploidie par FISH.

E-mail Address : sophie.rousseaux@ujf-grenoble.fr; International audience; Patients with severe spermatogenesis impairment can now successfully father a child thanks to the use of intracytoplasmic sperm injection (ICSI). In oligozoospermic patients, many studies have reported significantly higher sperm aneuploidy rates and therefore an increased risk of transmitting a chromosomal abnormality via the injection of abnormal spermatozoa. However, the frequency of aneuploidy is highly variable between patients. The aim of the present work was to identify clinical and biological factors, which, together with non-obstructive oligozoospermia, could be predictive of elevated sperm aneuploidies. The sperm aneuploidy rates for chromosomes X, Y, 13, 18 and 21 were assessed in 31 infertile men with well-characterized spermatogenesis impairment, and in a population of control men with proven fertility. The frequency of sperm aneuploidy was compared between several patient subgroups according to their clinical and biological factors. Nearly half of the oligozoospermic males (15/31) had a significantly increased disomy rate for at least one of the five chromosomes compared with that observed in the control population (mean disomy rates + 1.96 standard deviation). Factors significantly associated with higher numbers of aneuploid sperm were cigarette smoking, an elevated follicle-stimulating hormone level, a sperm concentration less than 1 M/mL, and a severe teratozoospermia. Hence, several factors predictive of an increased risk of sperm aneuploidy rates were identified in ICSI male candidates with a non-obstructive oligozoospermia.

International audience; We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia maybe associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in the companion article that oocyte activation (OA) with globozoospermic sperm is very poor and due to the absence of phospholipase C zeta; therefore artificial OA (AOA) was used to bypass defective OA. Herein, we evaluated the developmental potential of embryos generated by ICSI + AOA in mice. We demonstrate that although OA was fully rescued, preimplantation development was impaired when using globozoospermic sperm. In human, a small number of embryos could be generated with sperm from DPY19L2-deleted patients in the absence of AOA and these embryos also showed a poor developmental potential. In conclusion, we show that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Most of the DNA breaks were already present when the sperm reached the epididymis, indicating that they occurred inside the testis. This result thus suggests that testicular sperm extraction in Dpy19l2-dependent globozoospermia is not recommended. These defects may largely explain the poor embryonic development of most mouse and human embryos obtained with globozoospermic sperm.

In addition to genetic information, the spermatozoon carries another type of information, named epigenetic, which is not associated with variations of the DNA sequence. In somatic cells, it is now generally admitted that epigenetic information is not only regulated by DNA methylation but also involves modifications of the genome structure, or epigenome. During male germ cell maturation, the epigenome is globally re-organized, since most histones, which are associated to DNA in somatic cells, are removed and replaced by sperm specific nuclear proteins, the protamines, responsible for the tight compaction of the sperm DNA. However, a small proportion of histones, and probably other proteins, are retained within the sperm nucleus, and the structure of the sperm genome is actually heterogeneous. This heterogeneity of the sperm epigenome could support an epigenetic information, transmitted to the embryo, which could be crucial for its development. Although it is nowadays possible to appreciate the global structure of the sperm genome, the precise constitution of the sperm epigenome remains unknown. In particular, very recent data suggest that specific regions of the genome could be associated with particular proteins and define specific structures. This structural partitioning of the sperm genome could convey important epigenetic information, crucial for the embryo development.

L’activite d’autoconservation de sperme est devenue une activite de premier plan dans les CECOS. En 2002, 2323 patients ont ete adresses aux CECOS pour congelation et conservation de leurs spermatozoides avant radio ou chimiotherapie. La congelation d’un ou plusieurs recueils a pu etre realisee pour 2124 patients, ce qui represente un total de plus de 60000 paillettes sur une annee. Parmi les autoconservations avant traitement a risque sterilisant, les deux pathologies principalement concernees sont les cancers testiculaires (environ 40% des demandes) et les lymphomes (environ 30% des demandes). Il s’agit en general de conservations sur plusieurs annees. Du fait de l’amelioration continuelle des traitements, le pronostic de fertilite ulterieure des patients s’est ameliore ces dernieres annees, mais reste souvent difficile voire impossible a predire. Une appreciation de l’utilisation effective de ces gametes congeles et des resultats obtenus semblait donc interessante. Durant l’annee 2002, 304 hommes ont demande l’utilisation de leurs spermatozoides cryoconserves. Plus de 1000 paillettes ont ainsi ete decongelees et utilisees en inseminations (195 cycles d’inseminations, 12 grossesses obtenues) ou en fecondationin vitro (25 cycles de FIV conventionnelle, 8 grossesses; 257 cycles de FIV-ICSI, 57 grossesses). Une etude retrospective cumulative menee en 2001 avec la collaboration de 15 des 23 CECOS a permis de calculer pour chaque annee de congelation, parmi les patients ayant beneficie d’une cryoconservation, le pourcentage de patients qui ont demande l’utilisation des paillettes entre la date de debut de conservation et 2001. Ce pourcentage varie entre 5 et 10%, selon le delai ecoule depuis la congelation. Un calcul du pourcentage de patients pour lesquels la destruction des paillettes avait ete realisee, soit a la demande du patient, soit en raison du deces de celui-ci a egalement ete fait selon la meme methodologie. Le pourcentage de destructions pour deces du patient varie entre 5 et 9%. Le pourcentage de destructions des paillettes a la demande des patients avoisine ou depasse 15%. des lors que le delai depuis la congelation depasse 5 ans. Dans ces indications de cryoconservation, le pourcentage de patients perdus de vue reste faible, variant entre 3 et 6% selon les annees. Ces donnees sont globalement coherentes avec les quelques donnees bibliographiques disponibles. Meme si l’utilisation des paillettes n’est pas le devenir le plus frequent des cryoconservations de sperme, une congelation de gametes doit cependant toujours etre proposee aux patients car le devenir de fertilite des patients demeure souvent difficile a prevoir. Par ailleurs, l’evolution des techniques d’aide medicale a la procreation permet d’esperer une grossesse meme si peu de spermatozoides vivants ont pu etre conserves.

Purpose: Lymphomas and testicular cancers are the most frequent malignancies among young men. With recent improvement of survival rates, for many patients, the question is raised of the consequences of the anticancer treatments on their fertility and more specifically of a potential genetic risk for the offspring. This article presents the study of sperm aneuploidy rates in the largest population of cancer-treated patients studied thus far.Experimental Design: In the present study, 38 patients were initially included 7 months to 5 years after a cancer treatment by chemotherapy and/or radiotherapy for testicular cancer (n = 19) or lymphoma (n = 19). Twelve of them were azoospermic. Sperm aneuploidy rates of chromosomes X, Y, 13, 18, and 21 were analyzed by multicolor fluorescent in situ hybridization in the 26 other patients.Results: In most cases, the disomy/diploidy rates after cancer therapy did not significantly differ from those observed in the group of control healthy donors. Only five patients (one lymphoma and four testicular cancer) showed significant but still moderate increases in disomic and/or diploid sperm. For the lymphoma patient, the short posttherapeutic delay after the treatment could explain the elevated aneuploidy rates, whereas no risk factor in the clinical, biological, or therapeutic records could be identified in any of the four testicular cancer patients with elevated sperm aneuploidy rates.Conclusions: These data suggest an absence of long-term effect of anticancer therapy on sperm aneuploidy rates, and therefore, no long-term increased risk of aneuploidy for the offspring obtained either spontaneously or after assisted reproductive techniques.

Improvement of healthcare quality and safety are two main goals for hospitals. High risk preparations of injectable drugs is one of the possible areas for improvement. In this context the production of batches of standard doses is a practical solution in response to the increased demand. Some toolkits exists to facilitate the implementation of dose banding, but, to our knowledge, no complete strategy was available until today.To propose a rational approach to analyse the possibility of implementing standard doses and choose the most relevant drugs for dose standardization.The method is based on the analysis of literature focusing on different themes: safety, international guidelines, batch production regulation and stability studies.An approach on the strategies to develop is detailed for pharmacists willing to implement standard doses. All key stages are discussed: the needs of care units, the analysis and risk assessment, the stability studies and the practical implementation of the standard doses preparation and quality control.The implementation of standard doses seems a rational and necessary evolution of hospital pharmacy in response to the increase of compounding activity and the requirements of quality preparation. A global and all-inclusive approach is needed for this purpose. All parameters have to be considered to avoid errors. A process and a decision aid are suggested to facilitate the development of standard doses.

International audience; Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Intracytoplasmic Sperm Injection (ICSI) is an Assisted Reproduction Technique (ART) which offers the chance to conceive to patients presenting very low sperm counts (cryptozoospermia/severe oligozoospermia). Sperm freezing before the oocyte pick-up, can prevent from a lack of spermatozoa on the day of the ICSI. It can avoid the cancellation of the ICSI or the use of TESE (Testicular sperm extraction). The objective of this study was to analyse the practice of sperm freezing for these patients in our center over 8 years and the rate of use of these frozen sperms. We also compared the outcome of ICSIs with frozen versus ejaculated sperm.We performed a retrospective epidemiological study between 2004 and 2011. We recruited all the patients having a sperm count below 1 Million/mL and who were waiting for their first ICSI attempt.169 patients were recruited: 84 cryopreserved their sperm before the ICSI (secured ICSI) while 85 did not (non-secured ICSI). Both groups were split in cryptozoospermia (10(3) spermatozoa/ml): 19 and 17 patients respectively, very severe oligozoospermia (10(3)-10(5)/ml): 37 and 13 patients, and severe oligozoospermia (10(5)-10(6)/ml): 28 and 55 patients. The part of secured ICSI significantly increased from 29% during 2004-2007 to 74% during 2008-2011(p = 0.0029) and the frozen sperm was used in 5.9% of the cases. Median age was significantly higher in the non secured ICSI group (33.57 vs 35.52 for men, p = 0.0069 and 30.45 vs 32.26 for women, p = 0.025) but no significant difference was found in the outcome of the ICSI between frozen-thawed sperm and fresh ejaculated sperm.Sperm freezing before ICSI for severe oligozoospermic and cryptozoospermic patients significantly increased in our practice but the rate of use remain very low. This encourages to define more accurate criteria leading to sperm freezing.L’ICSI (Intracytoplasmic Sperm Injection) permet aux patients porteurs d’une oligozoospermie sévère ou d’une cryptozoospermie d’espérer concevoir un enfant. Ces patients courent parfois le risque de survenue d’une azoospermie constatée le jour de l’ICSI. L’organisation préalable d’une ou plusieurs congélations spermatiques peut permettre de prévenir ce risque. L’objectif de cette étude était d’évaluer cette pratique de congélation spermatique de sécurité sur une période prolongée dans notre centre et d’évaluer le taux d’utilisation de ces spermes congelés et les chances de grossesse au cours des ICSI, qu’elles soient faites avec les spermatozoïdes frais ou congelés et selon la gravité de l’oligozoospermie.Il s’agit d’une étude rétrospective monocentrique descriptive de 2004 à 2011. Les critères d’inclusion étaient une numération spermatique inférieure à 1 Million/mL pour une première tentative d’ICSI.169 patients ont été recrutés: 84 ont bénéficié de congélation de spermatozoïdes avant l’ICSI (ICSI sécurisée) et 85 patients n’en ont pas bénéficié (ICSI non sécurisée). Au sein de ces deux groupes, les patients ont été répartis en 3 sous-groupes en fonction de leur concentration spermatique: cryptozoospermie (10La congélation spermatique avant ICSI dans le cas des oligozoospermies sévères et cryptozoospermies est devenue presque systématique dans notre pratique. Pour autant l’utilisation de ces spermatozoïdes congelés demeure très peu fréquente. Ceci nous engage à poursuivre l’étude de ces situations afin de définir des critères cliniques et/ou biologiques justifiant ces congélations souvent itératives.

Mouse group X sPLA2 (mGX) is an acrosomal protein playing an important role in fertilization and controlling acrosome reaction (AR) occurring during capacitation. We demonstrated previously that sperm from mGX knock-out mice had a severely impaired fertilization potential in vitro. We also showed that treatment of wild-type sperm with recombinant mGX during capacitation improved fertilization outcome. This interesting property suggests that sPLA2s could be used to improve fertilization in assisted reproductive technologies (ART). However the molecular mechanism explaining the mGX-dependent enhancing effect on fertilization outcome remains unclear so far. Interestingly, like progesterone (P4), mGX is a very potent activator of AR and the role of mGX-induced AR in fertilization outcome was not evaluated so far. To assess the role of sPLA2-induced AR in IVF, we first tested the potency of 9 mouse and 2 human sPLA2s and P4 to trigger AR of mouse sperm. We then tested the ability of 6 of these molecules (mouse Group IIA, mouse Group IID, mouse Group X, human Group V, human Group X and P4) to improve the yield of 2-cell embryos obtained by IVF in mouse. We showed that in the mouse neither P4 nor any of the other sPLA2s tested were able to mimic the IVF improvement produced by mGX-treatment. These results demonstrate that sPLA2s are not commutable in the context of mouse sperm fertility, indicating that their utilisation in other species, is subjected to the identification of probably unique species-specific active sPLA2.

Parmi les anomalies cytogenetiques, le syndrome de Klinefelter est la plus frequente des anomalies chromosomiques associees a une infertilite masculine. Il concerne environ un nouveau-ne mâle sur 600. Sa frequence est de 3% dans la population des hommes infertiles et pres de 12% des sujets azoospermiques presentent un syndrome de Klinefelter. Il y a quelques annees, la seule possibilite de procreation pour ces couples etait de recourir au don de sperme. Depuis l’avenement des techniques de fecondationin vitro avec microinjection, il est possible d’obtenirin vitro une fecondation ovocytaire dans des cas d’oligozoospermie extreme. Par ailleurs, la possibilite de recueillir des spermatozoides chirurgicalement au niveau epididymaire ou testiculaire elargit les indications des infertilites pouvant beneficier d’une AMP intraconjugale. Ainsi, il est actuellement possible de proposer une AMP intraconjugale a certains couples, dont le conjoint est porteur d’un syndrome de Klinefelter, des lors que quelques spermatozoides mobiles peuvent etre obtenus, soit dans l’ejaculat, soit au niveau testiculaire. Cependant, le recours a ces techniques dans le cas d’une aneuploidie des cellules germinales pose le probleme de la transmission de cette aneuploidie a la descendance et du risque de sterilite de la descendance masculine. Ainsi, dans le cas du syndrome de Klinefelter, la transmission du chromosome X surnumeraire peut, en theorie, conduire a la naissance d’un garcon atteint de syndrome de Klinefelter ou d’une fille de caryotype 47XXX. Ce risque est directement lie au pourcentage de spermatozoides aneuploides 24XY ou 24XX. Ce pourcentage peut etre evalue actuellement par la technique d’hybridationin situ en fluorescence (Fluorescent In Situ Hybridization ou FISH) sur spermatozoides et des resultats peu nombreux et tres variables sont recenses dans la litterature. Nous presentons dans ce travail, les resultats obtenus pour un patient porteur d’un syndrome de Klinefelter a caryotype homogene et les donnees de la litterature concernant la segregation des chromosomes sexuels chez les patients proteurs de syndrome de Klinefelter. Dans le cas de notre patient, le taux de cellules aneuploides 24XY est environ dix fois plus eleve que le taux retrouve pour les sujets temoins. Par ailleurs, l’analyse des donnees bibliographiques, apporte des elements de reponses a quelques questions pratiques qui se posent lorsqu’on envisage une FIV avec microinjection chez ces patients: Par ailleurs, plusieurs naissances sont actuellement rapportees apres FIV avec microinjection pour des patients porteurs de syndrome de Klinefelter. Enfin, dans le cadre du conseil genetique, la place du diagnostic prenatal et/ou preimplantatoire est a discuter.

The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamineto human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.