Spermatozoa in mice lacking the nucleoporin NUP210L show defects in head shape and motility but not in nuclear compaction or histone replacement.

Biallelic loss-of-function mutation of NUP210L, encoding a testis-specific nucleoporin, has been reported in an infertile man whose spermatozoa show uncondensed heads and histone retention. Mice with a homozygous transgene intronic insertion in Nup210l were infertile but spermatozoa had condensed heads. Expression from this insertion allele is undefined, however, and residual NUP210L production could underlie the milder phenotype. To resolve this issue, we have created Nup210lem1Mjmm , a null allele of Nup210l, in the mouse. Nup210lem1Mjmm homozygotes show uniform mild anomalies of sperm head morphology and decreased motility, but nuclear compaction and histone removal appear unaffected. Thus, our mouse model does not support that NUP210L loss alone blocks spermatid nuclear compaction. Re-analyzing the patient's exome data, we identified a rare, potentially pathogenic, heterozygous variant in nucleoporin gene NUP153 (p.Pro485Leu), and showed that, in mouse and human, NUP210L and NUP153 colocalize at the caudal nuclear pole in elongating spermatids and spermatozoa. Unexpectedly, in round spermatids, NUP210L and NUP153 localisation differs between mouse (nucleoplasm) and human (nuclear periphery). Our data suggest two explanations for the increased phenotypic severity associated with NUP210L loss in human compared to mouse: a genetic variant in human NUP153 (p.Pro485Leu), and inter-species divergence in nuclear pore function in round spermatids.

Efficient pathway for men fertility preservation in testicular cancer or lymphoma: a cross-sectional study of national 2018 data.

BACKGROUND: In 15-49 years-old men, the main cancers are testicular cancer (TC) and lymphomas (L): freezing of ejaculated sperm is primarily used for male fertility preservation (FP) before cancer treatment. Our objective was to analyze the French FP rate in 15-49 years-old men diagnosed with TC or L in 2018. We designed a national descriptive cross-sectional study of sperm banking rate in men with a diagnosis of TC, Hodgkin L (HL) or non-Hodgkin L (NHL). From the French National Cancer Institute (INCa) 2018 data, we extracted the estimated incidence of TC and L in metropolitan France. From the 2018 activity report of CECOS network (Centers for Study and Banking of Eggs and Sperm), we extracted the number of men with TC or L who banked ejaculated sperm. We estimated the proportion of 15-49 years-old men diagnosed with TC or L who banked sperm. RESULTS: Among 15-49 years-old men, INCa estimated 38,048 new cancer diagnoses in metropolitan France in 2018: 2,630 TC and 3,913 L (943 HL and 2,970 NHL). The CECOS network provided data from 26/27 metropolitan centers (96% response rate): 1,079 sperm banking for men with TC, 375 for HL and 211 for NHL. We estimated that the 2018 sperm banking rate in France was 41% for TC, 40% for HL, and 7% for NHL. CONCLUSIONS: To our knowledge, our paper is the first cross-sectional study with multicenter and national data analyzing FP rate in cancer men: it suggests an efficient pathway for men to FP before cancer treatment, compared to previously published studies. Although sperm banking rate in 15-49 years-old men could definitely be improved, further studies should evaluate the information given to patients before gonadotoxic treatments, the factors associated with the absence of sperm banking and whether this lack of referral induces a loss of chance for these men.

RéSUMé: CONTEXTE: Chez les hommes de 15 à 49 ans, les principaux cancers sont le cancer du testicule (CT) et les lymhomes (L): la congélation de spermatozoïdes éjaculés est utilisée en première intention pour leur préservation de fertilité (PF) avant traitement du cancer. Notre objectif était d'analyser le taux de PF chez les hommes de 15 à 49 ans diagnostiqués avec un CT ou un L en 2018 en France. Nous avons réalisé une étude nationale transversale descriptive du taux de congelation de spermatozoïdes chez les hommes âgés de 15 à 49 ans diagnostiqués avec un CT, un L de Hodgkin (LH) ou un L non-Hodgkinien (LNH). A partir des données de l'Institut National du Cancer (INCa) de 2018, nous avons extrait l'incidence estimée de CT et de L en France métropolitaine. A partir des données du bilan d'activité 2018 de la Federation Française des CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme), nous avons extrait le nombre d'hommes avec un CT ou un L qui ont congelé leurs spermatozoïdes. Nous avons enfin estimé la proportion d'hommes de 15 à 49 ans diagnostiqués avec un CT ou un L qui ont congelé leurs spermatozoïdes. RéSULTATS: Chez les hommes de 15 à 49 ans, l'INCa a estimé en 2018 38 048 nouveaux cas de cancers diagnostiqués en France métropolitaine en 2018: 2 630 CT et 3 913 L (943 LH et 2 970 LNH). Le réseau des CECOS a produit les résultats issus de 26/27 centres métropolitains (taux de réponse de 96%): 1 079 congélations de sperme pour des hommes atteints de CT, 375 pour LH et 211 pour LNH. Nous avons estimé que le taux de congelation de spermatozoïdes de 2018 en France était de 41% pour le CT, 40% pour le LH et 7% pour le LNH. CONCLUSIONS: A notre connaissance, notre travail est la première étude transversale multicentrique de données nationales analysant le taux de PF chez les hommes atteints de cancer: il suggère un parcours patient efficace pour la PF des hommes avant traitement d'un cancer, par rapport aux études précédemment publiées. Bien que le taux de PF chez les hommes puisse certainemen être amélioré, des études futures devraient évaluer l'information donnée aux patients avant traitement gonadotoxique, les facteurs associés à l'absence de PF et si le défaut d'adressage au CECOS induit un perte de chance pour ces hommes. MOTS-CLéS: Chimiothérapie, Radiothérapie, Oncofertiité, Azoospermia, Paternité.

Feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation and childbearing desire after fertility-impairing treatment.

The aim of our study was to evaluate the feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation after fertility-impairing treatment regardless of the initial disease. We conducted a cohort study based on population of women < 40 years of age with diminished ovarian reserve caused by fertility-impairing treatment (n = 129). Three groups of women were compared according to the type of initial disease: hematological malignancies, solid tumors, and benign diseases. The primary endpoint was the number of metaphase II oocytes collected per woman. We studied the cumulative live-birth rate per cycle with fertilized metaphase II oocyte, for women who wanted to conceive. We studied 245 delayed controlled ovarian stimulation cycles in 129 women: 201 for fertility preservation and 44 for in vitro fertilization and fresh embryo transfers. The number of metaphase II oocytes collected per woman after banking was similar in the three groups, with a mean of 10.7 ± 4.6, 12.3 ± 9.1, and 10.1 ± 7.6 metaphase II oocytes (p = 0.46), respectively. In the subgroup of women who wanted to conceive, the cumulative live birth rate per woman was 38%, with 8 live births for these 21 women. After fertility-impairing treatment, practitioners should discuss a fertility preservation procedure for banking metaphase II oocytes.

Testicular germ cell tumour risk by occupation and industry: a French case-control study - TESTIS.

OBJECTIVE: Testicular germ cell tumours (TGCT) are the most common cancer in men of working age and its incidence has increased notably over the past 40 years. Several occupations have been identified as potentially associated with TGCT risk. The aim of this study was to further explore the relationship between occupations, industries and TGCT risk in men aged 18-45 years. METHODS: The TESTIS study is a multicenter case-control study conducted between January 2015 and April 2018 in 20 of 23 university hospital centers in metropolitan France. A total of 454 TGCT cases and 670 controls were included. Full job histories were collected. Occupations were coded according to the International Standard Classification of Occupation 1968 version (ISCO-1968) and industry according to the 1999 version of Nomenclature d'Activités Française (NAF-1999). For each job held, ORs and 95% CIs were estimated using conditional logistic regression. RESULTS: A positive association was observed between TGCT and occupation as agricultural, animal husbandry worker (ISCO: 6-2; OR 1.71; 95% CI (1.02 to 2.82)), as well as salesman (ISCO: 4-51; OR 1.84; 95% CI (1.20 to 2.82)). An increased risk was further observed among electrical fitters and related, electrical and electronics workers employed for 2 years or more (ISCO: 8-5; OR≥2 years 1.83; 95% CI (1.01 to 3.32)). Analyses by industry supported these findings. CONCLUSIONS: Our findings suggest that agricultural, electrical and electronics workers, and salesmen workers experience an increased risk of TGCT. Further research is needed to identify the agents or chemicals in these high-risk occupations which are relevant in the TGCT development. TRIAL REGISTRATION NUMBER: NCT02109926.

Subfertile patients underestimate their risk factors of reprotoxic exposure.

BACKGROUND: Exposure of men and women to environmental reprotoxic agents is associated with impaired fertility and pregnancy rates after assisted reproductive treatment (ART). Nevertheless, such exposures are generally not systematically assessed in current practice before ART and subfertile men are generally less explored than women. Our objective was to study subfertile men and women's level of knowledge about reprotoxic agents, their perception of their own risk factors and the correlation between perceived and identified circumstances of exposure. RESULTS: In our public university hospital, 390 subfertile patients (185 men and 185 women) requiring assisted reproduction technique (ART) treatment, completed a self-report questionnaire before consultation, in order to assess patients' knowledge of reprotoxic exposures, sources of information about them and perception of their own circumstances of exposure. Then a standardized questionnaire was used by the physician during the consultation to estimate domestic, environmental and occupational risk factors of reprotoxic exposures (RFRE). We compared the patients' perception of exposure with the estimated RFRE. The reprotoxic agents knowledge score of patients was 61%. Their main sources of information were the media (40%), the internet (22%) and gynecologists (15%). The standardized questionnaire identified RFRE in 265/390 patients (68%); risk factor was statistically more frequent in men (77%) than in women (59%) (p < 0.05). In total, 141 of the 265 patients with identified RFRE (53%) were aware of their risk factor of reprotoxic exposure. CONCLUSION: We identified risk factors of reprotoxic exposures in the majority of subfertile patients, more frequently in men than in women, and half of patients were not aware of their exposures. Patients' main sources of information were extra medical. Efforts should be made to inform patients, especially men, about potential reprotoxic exposure and to enhance medical training about reprotoxic agents, as recommended by international guidelines. The detection and correction of environmental exposures in subfertile men could improve their fecundity, but also their general health, which has been shown to be poorer than health of fertile men.

RéSUMé: CONTEXTE: L'exposition des hommes et des femmes à des agents reprotoxiques environnementaux est. associée à une atération de leur fertilité et des taux de grossesse après assistance médicale à la procreation (AMP). Néanmoins, ces expositions ne sont généralement pas recherchées en pratique courante avant AMP et les hommes infertiles sont généralement moins explorés que les femmes. Notre objectif était d'analyser le niveau de connaissance des hommes et des femmes infertiles sur les expositions environnementales reprotoxiques, leur perception de leurs propres facteurs de risque et la correlation entre les expositions reprotoxiques perçues et celles identifiées. RESULTATS: Dans notre centre hospitalier universitaire, 390 patients infertiles (185 hommes et 185 femmes) nécessitant un traitement d'AMP ont complété un auto-questionnaire avant la consultation, afin d'évaluer leurs connaissances sur les expositions reprotoxiques, leurs sources d'information sur ce sujet, et leur perception de leurs propres expositions. Puis, lors de la consultation, le médecin utilisait un questionnaire standardisé pour estimer leurs facteurs de risque d'exposition reprotoxique (FRER) domestiques, environnementaux et professionnels. Nous avons comparé la perception par les patients de leurs propres expositions reprotoxiques avec le FRER estimé par le médecin. Le score de connaissance des agents reprotoxique des patients était de 61%. Leurs sources d'information principales étaient les medias (40%), internet (22%), et les gynécologues (15%). Le questionnaire standardisé identifiait un FRER chez 265/390 patients (68%); les FRER étaient significativement plus fréquents chez les hommes (77%) que chez le femmes (59%)(p < 0.05). Au total, 141 patients sur les 265 avec un FRER identifié étaient conscients de leur FRER. CONCLUSION: Nous avons identifié des facteurs de risque d'exposition reprotoxiques chez la majorité des patients infertiles, plus fréquemment chez les hommes que chez les femmes, et la moitié des patients n'étaient conscients de ces expositions. Les principals sources d'information des patients étaient extra-médicales. Des efforts sont nécessaires pour informer les patients, en particulier les hommes, sur les potentielles expositions reprotoxiques comme souligné par les recommandations internationales. La detection et la correction des expositions environnementales chez les hommes infertiles pourraient améliorer leur fécondité, mais aussi leur santé, qui a été démontrée comme moins bonne que celle des hommes fertiles.

Fertility preservation and sperm donation in transgender individuals: The current situation within the French CECOS network.

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 < 0.0001) and achieve (p < 0.0001) fertility preservation than transgender men. Concerning sperm donation in couples including a transgender man, 68% of centres offer the complete course from the first consultation to the completion of the assisted reproductive technology cycles; 122 offsprings have been conceived with sperm donation in couples including a transgender man since 1999. DISCUSSION: Our results showed that even if all centres do not propose fertility preservation or sperm donation in transgender individuals, these assisted reproductive technologies are present throughout the French territory. The major point is that both fertility preservation and sperm donation in transgender individuals have grown significantly and that the care of these patients is improving year after year. CONCLUSION: In France, most of CECOS centres can take care of transgender individuals for fertility preservation and sperm donation. The French Bioethics Law allows these latter, and transgender individuals can benefit from a financial support of the national health care insurance for fertility preservation and sperm donation.

Genetic analyses of a large cohort of infertile patients with globozoospermia, DPY19L2 still the main actor, GGN confirmed as a guest player.

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.

Fertility preservation in Turner syndrome: Karyotype does not predict ovarian response to stimulation.

OBJECTIVE: Turner syndrome (TS) is responsible for gonadal dysgenesis with high risk of premature ovarian insufficiency. Little is known about fertility preservation (FP) strategies is this population. DESIGN: Data from women with TS consulting with a fertility specialist in our FP centre from 2014 to 2018 were retrospectively collected. MEASUREMENT: Total number of mature oocytes cryopreserved using vitrification. PATIENTS: Nine women with TS were referred. Three women with different karyotypes underwent controlled ovarian stimulation (COS) for oocyte vitrification. Mean age at TS diagnosis was 13.7 years [9-20]. Mean referral delay between TS diagnosis and fertility consultation was 9.7 years [7-14]. First counselling for FP was provided at 23.7 years [18-28]. Mean AMH serum level prior to COS was 53.8 pmol/L [3.6-95]. RESULTS: All three women succeeded in obtaining cryopreserved oocytes with a mean number of 15.3 per woman [9-20] and 9.2 per COS cycle [2-20]. Ovarian response to COS was unexpectedly remarkable for the woman with a complete 45,X monosomy. Procedure was well tolerated for all women. None of them have used oocytes for in vitro fertilization yet. CONCLUSIONS: Independently of karyotype, antral follicular count, AMH and FSH levels seemed to be reliable predictive markers of oocyte cryopreservation success. In a monosomic TS woman, cryptic ovarian mosaicism could explain a successful ovarian response to stimulation with a high number of retrieved oocytes. In case of spontaneous menarche, TS adolescents should be referred during transition to adulthood for FP counselling to avoid referral delay and limit time-related diminished ovarian reserve.

Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men.

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs∗4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111∗) and chr12: g.122395032_122395033delCT (p.Leu530Valfs∗4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.

The involvement of the nuclear lamina in human and rodent spermiogenesis: a systematic review.

The nuclear lamina (NL) is a filamentous protein meshwork, composed essentially of lamins, situated between the inner nuclear membrane and the chromatin. The NL is a component of the nuclear envelope, interacts with a wide range of proteins and is required for normal nuclear structure and physiological development. During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. There is mounting evidence from studies in human and rodent, that the NL plays an important role in mammalian spermatid differentiation during spermiogenesis. In this review, we summarize and discuss the data available in the literature regarding the involvement of lamins and their direct or indirect partners in normal and abnormal human spermiogenesis.

La lamina nucléaire (LN) est un réseau de filaments protéiques, composé essentiellement de lamines, situé entre la membrane nucléaire interne et la chromatine. La LN est un composant de l'enveloppe nucléaire, interagit avec une large gamme de protéines et est nécessaire à l'intégrité de la structure nucléaire et au développement physiologique. Au cours de la spermiogenèse, le noyau de la spermatide s'allonge et sa taille est considérablement réduite, les protamines remplaçant les histones dans le but de constituer une chromatine fortement compactée. De nombreux travaux chez l'homme et chez les rongeurs montrent que la LN joue un rôle important dans la différenciation des spermatides chez les mammifères au cours de la spermiogenèse. Dans cette revue, nous résumons et discutons les données disponibles dans la littérature concernant l'implication des lamines et de leurs partenaires directs ou indirects dans la spermiogenèse humaine normale et anormale.

Donor sperm insemination after failed intra-couple intracytoplasmic sperm injection.

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. ABBREVIATIONS: 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; ß hCG: beta human chorionic gonadotrophin; SD: standard deviation; OR: Odds ratio.

Abnormal retention of nuclear lamina and disorganization of chromatin-related proteins in spermatozoa from DPY19L2-deleted globozoospermic patients.

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, LAP2α, BAF and BAF-L, by reverse transcriptase-PCR 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, LAP2α 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.

Sperm donor conception and disclosure to children: a 10-year retrospective follow-up study of parental attitudes in one French center for the study and preservation of eggs and sperm (CECOS).

OBJECTIVE: To evaluate the percentage of parents in one French center for the study and preservation of eggs and sperm who disclose their use of donated spermatozoa to their children. DESIGN: A questionnaire survey of couples who had a child using donated spermatozoa. SETTING: University hospital laboratory. PATIENT(S): One hundred five couples. INTERVENTION(S): Questionnaire sent by mail. MAIN OUTCOME MEASURE(S): The percentage of parents who disclose their use of donated spermatozoa to their child. RESULT(S): Among the 157 questionnaires sent, 105 couples answered, which corresponded to 138 children. There were 40 (38%) couples who had already disclosed the donor origin to their child and 65 (62%) who had not. Of the 40 couples who disclosed the donor origin, 37 (93%) had intended to do so before making use of assisted reproductive techniques (ART), but two (5%) had not wanted to do so before ART. Among the 65 couples who did not inform their child, 42 (65%) planned to inform their child soon, but 20 (31%) wanted to keep the sperm origin secret. Of the 20 couples who wanted to keep the origin secret, nine couples had told other persons about the gamete donation but had not informed their child and do not intend to inform their child in the future. CONCLUSION(S): This first report about disclosure attitude in a large cohort of parents of donor-conceived offspring in France showed that most parents had already disclosed their use of donated spermatozoa to their children or intended to disclose it soon and had an attitude after birth consistent with their intentions prior to ART.

LEM-domain proteins are lost during human spermiogenesis but BAF and BAF-L persist.

During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. After fertilisation, this process is reversed in the oocyte to form the male pronucleus. Emerging evidence, including the coordinated loss of the nuclear lamina (NL) and the histones, supports the involvement of the NL in spermatid nuclear remodelling, but how the NL links to the chromatin is not known. In somatic cells, interactions between the NL and the chromatin have been demonstrated: LEM-domain proteins and LBR interact with the NL and respectively, the chromatin proteins BAF and HP1. We therefore sought to characterise the lamina-chromatin interface during spermiogenesis, by investigating the localisation of six LEM-domain proteins, two BAF proteins and LBR, in human spermatids and spermatozoa. Using RT-PCR, IF and western blotting, we show that six of the proteins tested are present in spermatids: LEMD1, LEMD2 (a short isoform), ANKLE2, LAP2ß, BAF and BAF-L, and three absent: Emerin, LBR and LEMD3. The full-length LEMD2 isoform, required for nuclear integrity in somatic cells, is absent. In spermatids, no protein localised to the nuclear periphery, but five were nucleoplasmic, receding towards the posterior nuclear pole as spermatids matured. Our study therefore establishes that the lamina-chromatin interface in human spermatids is radically distinct from that defined in somatic cells. In ejaculated spermatozoa, we detected only BAF and BAF-L, suggesting that they might contribute to the shaping of the spermatozoon nucleus and, after fertilisation, its transition to the male pronucleus.

Homozygous deletion of SUN5 in three men with decapitated spermatozoa.

A recent study of 17 men with decapitated spermatozoa found that 8 carried two rare SUN5 alleles, and concluded that loss of SUN5 function causes the acephalic spermatozoa syndrome. Consistent with this, the SUN5 protein localises to the head-tail junction in normal spermatozoa, and SUN proteins are known to form links between the cytoskeleton and the nucleus. However, six of the ten SUN5 variants reported were missense with an unknown effect on function, and only one man carried two high confidence loss-of-function (LOF) alleles: p.Ser284* homozygozity. One potential exonic splice mutation, homozygous variant p.Gly114Arg, was not tested experimentally. Thus, definitive proof that loss of SUN5 function causes the acephalic spermatozoa syndrome is still lacking. Based on these findings, we determined the sequence of the SUN5 gene in three related men of North African origin with decapitated spermatozoa. We found all three men to be homozygous for a deletion-insertion variant (GRCh38 - chr20:32995761_32990672delinsTGGT) that removes 5090 base pairs including exon 8 of SUN5, predicting the frameshift, p.(Leu143Serfs*30), and the inactivation of SUN5. We therefore present the second case where the acephalic spermatozoa syndrome is associated with two LOF alleles of SUN5. We also show that the p.Gly114Arg variant has a strong inhibitory effect on splicing in HeLa cells, evidence that homozygozity for p.Gly114Arg causes acephalic spermatozoa syndrome through loss of SUN5 function. Our results, together with those of the previous study, show that SUN5 is required for the formation of the sperm head-tail junction and male fertility.

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes.

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.

Sperm mRNAs and microRNAs as candidate markers for the impact of toxicants on human spermatogenesis: an application to tobacco smoking.

Spermatozoa contain a complex population of RNAs including messenger RNAs (mRNAs) and small RNAs such as microRNAs (miRNA). It has been reported that these RNAs can be used to understand the mechanisms by which toxicological exposure affects spermatogenesis. The aim of our study was to compare mRNA and miRNA profiles in spermatozoa from eight smokers and eight non-smokers, and search for potential relationships between mRNA and miRNA variation. All men were selected based on their answers to a standard toxic exposure questionnaire, and sperm parameters. Using mRNA and miRNA microarrays, we showed that mRNAs from 15 genes were differentially represented between smokers and non-smokers (p<0.01): five had higher levels and 10 lower levels in the smokers. For the microRNAs, 23 were differentially represented: 16 were higher and seven lower in the smokers (0.004≤p<0.01). Quantitative RT-PCR confirmed the lower levels in smokers compared to non-smokers for hsa-miR-296-5p, hsa-miR-3940, and hsa-miR-520d-3p. Moreover, we observed an inverse relationship between the levels of microRNAs and six potential target mRNAs (B3GAT3, HNRNPL, OASL, ODZ3, CNGB1, and PKD2). Our results indicate that alterations in the level of a small number of microRNAs in response to smoking may contribute to changes in mRNA expression in smokers. We conclude that large-scale analysis of spermatozoa RNAs can be used to help understand the mechanisms by which human spermatogenesis responds to toxic substances including those in tobacco smoke.