Virginie Barraud-Lange

Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin.

Microinjection of nuclear vacuole-free spermatozoa selected by motile sperm organellar morphological examination (MSOME) has been claimed to enhance assisted reproduction treatment outcome compared with intracytoplasmic sperm injection. However, the nature of these nuclear vacuoles is unclear, since their localization at the front of the sperm head suggests they might be of acrosomal origin. To study this hypothesis, acrosomal status was evaluated using Pisum sativum agglutinin staining on a smear, together with sperm organellar morphological examination using the same optics as for MSOME on 30 sperm samples from infertile patients, yielding >3200 spermatozoa. Vacuoles were present in 61% of spermatozoa when acrosomal material or intact acrosomes were observed, versus 29% when spermatozoa were acrosome reacted (P<0.0001). Induction of the acrosomal reaction by ionophore A23587 from 17.4% to 36.1% significantly increased the percentage of vacuole-free spermatozoa from 41.2% to 63.8% (P<0.001). These data suggest that most nuclear vacuoles are of acrosomal origin. Hence, the best spermatozoa selected by MSOME are mostly acrosome-reacted spermatozoa. As microinjection of spermatozoa with a persistent acrosome drastically hampers embryo development in animal models, this suggests that the improvement in pregnancy rates reported following intracytoplasmic injection of morphologically selected sperm might be due to the procedure allowing injection of acrosome-reacted spermatozoa.

Seminal leukocytes are Good Samaritans for spermatozoa

OBJECTIVEnTo assess the effect of leukocytospermia on assisted reproductive technology outcomes.nnnDESIGNnRetrospective analysis.nnnSETTINGnUniversity laboratory.nnnPATIENT(S)nCouples attending the infertiliy clinic and involved in ART program for IVF or ICSI.nnnINTERVENTION(S)nDuring a 7-year follow-up in an assisted reproductive technology program, leukocytospermia was routinely determined using the peroxidase technique. Donor sperm were excluded from the study.nnnMAIN OUTCOME MEASURE(S)nEgg retrievals (N = 3,508) were distributed in 3 groups according to the leukocyte levels in semen from which fertilizing sperm were extracted: group 1, absence of leukocytes (n = 3,026); group 2, moderate leukocytospermia (<10(6)/mL) (n = 344); or group 3, high leukocytospermia (≥10(6)/mL) (n = 138). They resulted inxa01,463 IVF and 2,045 intracytoplasmic sperm injection procedures that gave 802 clinical pregnancies.nnnRESULT(S)nSurprisingly, the fertilization rate, cleavage rate, clinical pregnancy rate, gestational age, and mean infant weight were significantly improved when seminal leukocytes were present, regardless of the technique used. The only negative side effects associated with a high level of seminal leukocytes (group 3) were an elevated rate of early pregnancy loss (from 26.6% to 40.5%) and a 3-fold increase in the percentage of ectopic pregnancies.nnnCONCLUSION(S)nAt moderate levels (<10(6)/mL), leukocytospermia appears to be physiologic. It is associated with improved sperm fertilization ability and pregnancy outcome. At higher concentrations, leukocytospermia alters neither sperm fertilization ability nor the probability of clinical pregnancy when compared with nonleukocytic patients with infertility. However, the pregnancy outcome is reduced.

Short gamete co-incubation during in vitro fertilization decreases the fertilization rate and does not improve embryo quality: a prospective auto controlled study

PurposeEvaluate the effect of short gamete incubation on fertilization rate and embryo quality.MethodsA prospective study has been performed. Two thousand five hundred and forty seven sibling oocytes from 240 couples undergoing IVF attempts were allocated to a short (1xa0h) or a standard (18xa0h) insemination procedure. Diploid fertilization rate (two pronuclei, 2PN), polyspermy (>2PN) and embryo quality were compared.ResultsThe fertilization rate was statistically lower in the short insemination group compared to the standard insemination one (64.9% and 70.1%; Pu2009=u20090.039), with a similar polyspermy rate observed between the two groups. A slight, but non significant, increase was observed concerning good embryo quality rate in the short insemination group when compared to the standard insemination, both at day 2 (60.1 vs. 58.1%; Pu2009=u20090.06) and day 3 (53.2 vs. 48.5%; Pu2009=u20090.22).ConclusionThis new study highlights that a 1xa0h gamete exposure decreases the fertilization rate and does not improve embryo quality compared with a standard 18xa0h insemination procedure.

Cyclic QDE peptide increases fertilization rates and provides healthy pups in mouse.

OBJECTIVEnTo evaluate the effects of cyclic QDE peptide (cQDE), derived from sperm fertilin beta (ADAM2), in mouse in vitro fertilization (IVF) and its harmlessness on pups after embryo transfer.nnnDESIGNnProspective in vivo and in vitro study in mice.nnnSETTINGnMurine model in an academic research environment in France.nnnANIMAL(S)nNormal B6CBF1 mice.nnnINTERVENTION(S)nIndirect immunofluorescence was used to evaluate cQDE binding to oolemma. The IVF assays were run in presence of the species-specific tripeptide (cQDE at 100 microM), and embryos were transferred in pseudopregnant mice. Controls were obtained by natural mating and IVF in regular medium followed by embryo transfer.nnnMAIN OUTCOME MEASURE(S)nEvaluation of fertilization, litter size, pup fertility and health.nnnRESULT(S)nBiotinylated cQDE peptide binds to oocyte plasma membrane and increases gamete fusion in cumulus-intact IVF assay. Litter size as well as pup development after embryo transfer showed no statistically significant differences compared with controls. Pups born from embryos that were incubated with the peptide were as healthy and normally fertile as control mice over at least three generations.nnnCONCLUSION(S)nCyclic QDE is harmless and improves mouse IVF pregnancy rates. A randomized prospective clinical trial to evaluate the beneficial effect of cyclic FEE on human IVF is required before its clinical use.

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

STUDY QUESTIONnThe aim of this study was to evaluate the live birth rate (LBR) after frozen-thawed Day 5 (D5) and Day 6 (D6) blastocyst transfers.nnnSUMMARY ANSWERnLBR following frozen-thawed blastocyst transfer is significantly lower with D6 than with D5 blastocyst regardless of embryo quality.nnnWHAT IS KNOWN ALREADYnDuring fresh embryo transfer cycles, pregnancy rates (PR) are significantly higher when transferring blastocysts expanded on D5 compared with slow developing blastocysts (D6). In programmed thawed blastocyst transfer (TBT) cycles, the same clinical outcomes should be expected when transferring D5 or D6 blastocysts because of endometrial/embryonic synchronization due to hormonal priming of endometrial receptivity. However, the impact of delayed blastocyst expansion at D6 on clinical outcomes remains unclear. Some reports have shown higher PRs after D5 TBT compared with those of D6, while others have shown equivalent TBT outcomes after D5 and D6 cryopreserved blastocysts transfers.nnnSTUDY, DESIGN, SIZE, DURATIONnThis retrospective cohort follow-up study included 1347 single autologous frozen-thawed blastocyst transfers performed between January 2012 and December 2015 at a tertiary care university hospital.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnAll of the patients scheduled for TBT were allocated to two groups according to the day of blastocyst expansion: on D5 (n = 994) or on D6 (n = 353). The primary outcome was LBR per embryo transfer in the first blastocyst thawing cycle. Secondary outcomes were clinical pregnancy rate (cPR), early miscarriage rate and neonatal outcomes following TBT for the two groups. Statistical analyses were conducted using univariate and multivariate logistic regression model.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe LBR was significantly increased in the D5 group compared to the D6 group [294/994 (29.6%) versus 60/353 (17.0%); P < 0.001]. The cPR was also higher when blastocysts were vitrified on D5 compared with those vitrified on D6 [429/994 (43.2%) versus 95/353 (26.9%); P < 0.001]. No significant differences were found between groups in terms of early miscarriage rate (P = 0.862). More good-quality embryos (defined as an B3-B4 or B5 embryo ≥BB according to the grading scale proposed by Gardner) were transferred in the D5 group than in the D6 group [807 (81.2%) versus 214 (60.6%); P < 0.001]. However, a comparison of TBT cycles with equal embryo quality (good versus low) also supported the superiority of D5 blastocysts. Concerning neonatal outcomes, the D5 group infants had a lower mean birth weight compared to those of the D6 group (P = 0.001). In addition, a significantly shorter gestational age at birth is reported in the D5 blastocyst group as compared to the D6 group (P = 0.004). After multivariate logistic regression taking into account potential confounders such as the womens age, number of previous IVF/ICSI procedures, the day of the blastocyst vitrification (D5 or D6) and embryo quality, blastocyst expansion at D6 was independently associated with a significant decrease in LBR compared to D5 expanded-blastocysts (OR 0.52; 95% CI 0.38-0.72; P < 0.001).nnnLIMITATIONS, REASONS FOR CAUTIONnThe poor predictive value of the morphological approach in embryo selection could constitute a limitation in this study. However, blastocyst quality was evaluated similarly in both groups.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe LBR following frozen-thawed blastocyst transfer was significantly lower with D6 than with D5 blastocysts, regardless of their quality. These results could affect cryopreservation procedures as they suggest that the use of D5-expanded blastocysts for TBT may be preferred in order to shorten the time of conceiving.nnnSTUDY FUNDING/COMPETING INTEREST(S)nNo specific funding was obtained for this study. None of the authors have any competing interests to declare.nnnTRIAL REGISTRATION NUMBERnNot applicable.

Extended culture of poor-quality supernumerary embryos improves ART outcomes

PurposeThe aims of this study were to investigate the possible benefits of extending the culture of poor-quality day-2 embryos (PQE) versus good-quality embryos (GQE) and to identify factors associated with pregnancy and live birth when transferring frozen-thawed blastocysts originating from GQE and PQE.MethodsThis is a retrospective cohort follow-up study performed between November 2012 and February 2015 at the IVF Laboratory Unit of Cochin University Hospital (Paris, France) including 3108 day-2 supernumerary embryos resulting from 1237 IVF/ICSI cycles.ResultsTotal blastulation rate was 67.2% from GQE and 48.7% from PQE. Percentage of good-quality blastocysts was 60.7 and 47.9% respectively including 14.7 and 7.3% top-quality blastocysts. A total of 150 blastocysts originating from GQE and 729 from PQE were frozen, and then, 37 and 164 were thawed and transferred respectively resulting in 19 (51.4%) and 61 (37.9%) clinical pregnancies with 13 (35.1%) deliveries from GQE and 32 (19.9%) from PQE (pxa0=xa00.046) without any difference in neonatal outcomes. Quality of blastocysts that resulted in live birth was similar in the two groups. Women <xa035xa0years old and day-5 blastocyst expansion were predictive of pregnancy and live birth.Conclusions(i) PQE are able to reach the blastocyst stage, to implant, and to give healthy babies and (ii) women age and day of blastocyst expansion are predictive of pregnancy and live birth.

L’interaction gamétique au cours de la fécondation

L’ovocyte qui va etre feconde a grandi en taille au sein de son follicule et s’est entoure de la zone pellucide, structure amorphe acellulaire composee de glycoproteines au nombre de quatre dans l’espece humaine (1). Cette zone pellucide va avoir plusieurs fonctions: la reconnaissance et la selection du spermatozoide fecondant, ainsi que le declenchement de sa reaction acrosomique lui permettant de liberer les enzymes qu’il transporte dans l’acrosome. Ceux-ci vont lui permettre de rompre les liaisons entre la ZP1 et les polymeres de ZP2-ZP3 afi n de se frayer un passage et de se propulser au travers de la zone pellucide grâce a ses battements flagellaires jusque dans l’espace perivitellin (2).

L’intégrine α6β1 ovocytaire et spermatique dans l’interaction gamétique

RésuméDes tests d’inhibitions ont permis de proposer l’intégrine α6β1 comme le récepteur du spermatozoïde sur l’ovocyte mais des expériences d’invalidation de gène ont montré que les sous unités intégrines α6 et β1 ovocytaires n’étaient pas essentielles à la fécondation. En utilisant le Western blot et l’immunofluorescence (cytométrie de flux et microscopie), nous avons montré que I’α6β1 est exprimée par les spermatozoïdes de souris. Comme pour l’ovocyte, un anticorps anti-α6 (GoH3) inhibe spécifiquement les capacités fécondantes des spermatozoïdes.En comparant des tests de fusion avec des ovocytes intacts (avec zone pellucide) ou des ovocytes dépellucidés, nous avons montré que la dépellucidation court-circuite la fonction de l’α6β1 dans le processus d’adhésion/fusion des gamètes. L’α6β1 est donc exprimée et fonctionnelle sur les deux gamètes au cours de l’interaction de leur membranes.Nos résultats, ceux des expériences utilisant des ovocytes mutés systématiquement inséminés avec des spermatozoïdes de souris sauvages, ainsi que ceux des myoblastes qui sont incapables de fusionner entre eux lorsqu’ils sont mutés pour la sous-unité intégrine β1 alors qu’ils le font avec des myoblastes sauvages, nous conduisent à formuler l’hypothèse selon laquelle la présence de la sous-unité intégrine β1 sur une seule des deux membranes suffit pour que la fusion ait lieu. Cette hypothèse est renforcée par un phénomène d’échange de fragments membranaires entre les gamètes juste avant leur fusion que nous avons décrits tout récemment.AbstractBased on inhibition tests, the α6β1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte α6, nor β1 integrin subunits were essential for mouse fertilization. Using Western blot analysis and immunofluorescence (flow cytometry and microscopy), we have shown that mouse sperm expresses the α6β1 integrin.As for oocytes, binding of GoH3 anti-alpha6 antibody to sperm induces specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we have shown that removal of the zona pellucida bypasses the α6β1 integrin role in the adhesion/fusion process of oocyte fertilization. The α6β1 integrin is expressed by both gametes and is functional during their membrane interactions.Our results, previous reports on fertilization of α6 or β1 integrin subunit-deleted oocytes by wild-type sperm and the fusion ability of β1 mutant myoblasts when they were co-cultured with wild-type myoblasts suggest that the presence of α6βl integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the recently reported exchange of membrane fragments occurring between gametes prior to fusion.