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.

[Cryoconservation of gametes: how to perform?] / Conservation des gamètes : quelles modalités ?

Cryoconservation of gametes : how to perform ? The patients can preserve their gametes when they are exposed to potential gonadotoxic pathology or treatment. In this context, the French bioethical law clearly states the obligation to inform the patients about the risks for their fertility and the possibilities to cryopreserve their gametes. Regional platforms of fertility preservation allow notably for the coordination of the oncology teams and the CECOS. For the men, sperm freezing is achieved by a slow and controlled temperature protocol. For the women, the oocytes are usually vitrified after hormonal stimulation and ovarian punction. For both, the gametes are cryopreserved in straws and stored in liquid nitrogen until use in assisted reproductive treatment (ART). Each year, the CECOS keeping the gametes interrogates patients on their wish to continue, or not, the cryoconservation. The gametes can only be used in ART by the patients only during their lifetime and with their consent, without alterations related to the duration of storage.

Conservation des gamètes : quelles modalités ? Les patient(e)s peuvent conserver leurs gamètes lorsqu'ils( ou elles) sont exposé(e)s à une pathologie ou un traitement potentiellement gonadotoxique. Dans ce contexte, la loi de bioéthique énonce très clairement l'obligation d'informer les patients des risques pour leur fertilité et des possibilités de conservation de leurs gamètes. Les plateformes régionales de préservation de la fertilité assurent la coordination entre les équipes d'oncologie et les centres d'étude et de conservation des oeufs et du sperme humains (CECOS) pour la mise en oeuvre de cette préservation. Pour les hommes, la congélation des spermatozoïdes est réalisée par une descente en température lente et contrôlée. Pour les femmes, la cryoconservation des ovocytes se fait par vitrification après stimulation hormonale et ponction ovarienne. Dans les deux cas, les gamètes sont conservés dans des paillettes qui sont stockées dans une cuve d'azote liquide jusqu'à utilisation en assistance médicale à la procréation (AMP). Le CECOS conservant les gamètes interroge chaque année par courrier les patients concernés sur leur souhait de poursuivre, ou non, la cryoconservation. Les gamètes pourront être utilisés en AMP par les patients de leur vivant et après leur consentement, sans altération liée à la durée de stockage.

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.

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.

Nuclear envelope remodelling during human spermiogenesis involves somatic B-type lamins and a spermatid-specific B3 lamin isoform.

The nuclear lamina (NL) is a filamentous protein meshwork, composed essentially of lamins, situated between the inner nuclear membrane and the chromatin. There is mounting evidence that the NL plays a role in spermatid differentiation during spermiogenesis. The mouse spermatid NL is composed of the ubiquitous lamin B1 and the spermatid-specific lamin B3, an N-terminally truncated isoform of lamin B2. However, nothing is known about the NL in human spermatids. We therefore investigated the expression pattern and localization of A-type lamins (A, C and C2) and B-type lamins (B1, B2 and B3) during human spermiogenesis. Here, we show that a lamin B3 transcript is present in human spermatids and that B-type lamins are the only lamins detectable in human spermatids. We determine that, as shown for their mouse counterparts, human lamin B3, but not lamin B2, induces strong nuclear deformation, when ectopically expressed in HeLa cells. Co-immunofluorescence revealed that, in human spermatids, B-type lamins are present at the nuclear periphery, except in the region covered by the acrosome, and that as the spermatid matures the B-type lamins recede towards the posterior pole. Only lamin B1 remains detectable on 33-47% of ejaculated spermatozoa. On spermatozoa selected for normal head density, however, this fell to <6%, suggesting that loss of the NL signal may be linked to complete sperm nucleus compaction. The similarities revealed between lamin expression during human and rodent spermiogenesis, strengthen evidence that the NL and lamin B3 have conserved functions during the intense remodelling of the mammalian spermatid nucleus.

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.

Are men ready to use thermal male contraception? Acceptability in two French populations: New fathers and new providers.

BACKGROUND: Since the 1970s, international research has actively pursued hormonal male contraception (HMC) and, to a lesser extent, thermal male contraception (TMC). Although the efficacy of TMC has been confirmed in limited populations, its acceptability has not been studied in either potential users or potential prescribers. METHODS: A cross-sectional descriptive multicentre study of potential male users of TMC (new fathers) and potential prescribers of TMC (new providers) was conducted between November 2016 and February 2017.The participants completed a 3-part survey, and their responses were evaluated to i) determine their socio-demographic profiles; ii) identify personal experiences with contraception; and iii) gauge the participants' knowledge, interest and preference for male contraception, particularly TMC. For new providers only, the survey included a fourth part to evaluate professional experience with male contraception. RESULTS: The participation rate was 51% for new fathers (305 NFs) and 34% for new providers (300 NPs, including 97 men (male new providers, MNPs) and 203 women (female new providers, FNPs)). Only 3% of NFs and 15% of NPs knew about TMC (including 26% of the MNPs and 10% of the FNPs, p<0.01). After reading information on TMC, new fathers were significantly less willing to try TMC (29%) than were new providers (40%) (p<0.01). The 3 main advantages of TMC for the new fathers included the following factors: "natural" (52%), "without side effects" (38%) and "non-hormonal" (36%). The main disadvantages were "lengthy wear time" (56%), "daily undergarment wear" (43%) and "concern about possible discomfort" (39%). CONCLUSIONS: Young male and female providers have limited knowledge of male contraception, are interested in further information and would generally prescribe TMC to their patients. Successful expansion of the use of male contraception, including TMC, would require distribution of better information to potential users and providers.

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.

Soluble CD146, an innovative and non-invasive biomarker of embryo selection for in vitro fertilization.

Although progress was made in in vitro fertilization (IVF) techniques, the majority of embryos transferred fail to implant. Morphology embryo scoring is the standard procedure for most of IVF centres for choosing the best embryo, but remains limited since even the embryos classified as "top quality" may not implant. As it has been shown that i) CD146 is involved in embryo implantation and ii) membrane form is shed to generate soluble CD146 (sCD146), we propose that sCD146 in embryo supernatants may constitute a new biomarker of embryo selection. Immunocytochemical staining showed expression of CD146 in early embryo stages and sCD146 was detected by ELISA and Western-blot in embryo supernatants from D2. We retrospectively studied 126 couples who underwent IVF attempt. The embryo culture medium from each transferred embryo (n = 222) was collected for measurement of sCD146 by ELISA. Significantly higher sCD146 concentrations were present in embryo supernatants that did not implant (n = 185) as compared to those that successfully implanted (n = 37) (1310 +/- 1152 pg.mL-1 vs. 845+/- 1173 pg.mL-1, p = 0.024). Sensitivity analysis performed on single embryo transfers (n = 71) confirmed this association (p = 0.0054). The computed ROC curve established that the optimal sCD146 concentration for embryo implantation is under 1164 pg.mL-1 (sensitivity: 76%, specificity: 48%, PPV: 25% and NPV: 92%). Over this sCD146 threshold, the implantation rate was significantly lower (9% with sCD146 levels >1164 pg.ml-1 vs. 22% with sCD146 levels ≤ 1164 pg.mL-1, p = 0.01). Among the embryos preselected by morphologic scoring, sCD146 determination could allow a better selection of the embryo(s), thus improving the success of elective single embryo transfer. This study establishes the proof of concept for the use of sCD146 as a biomarker for IVF by excluding the embryo with the highest sCD146 level. A multicentre prospective study will now be necessary to further establish its use in clinical practice.