Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality.

BACKGROUND: Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with ("H+" arm) or without ("H-" arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. RESULTS: Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and +  21.6% respectively, p < 0.005) than spermatozoa from the control "H-" arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (- 16.1%, p < 0.05), DNA fragmentation (- 18.7%, p < 0.05) and nuclear vacuolization (- 20.8%, p < 0.05). CONCLUSION: Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. TRIAL REGISTRATION: Clinical Trial, NCT04011813 . Registered 19 May 2019 - Retrospectively registered.

RéSUMé: CONTEXTE: Bien que largement utilisée, la congélation lente modifie considérablement les fonctions des spermatozoïdes humains. La cryoconservation induit des altérations nucléaires du sperme et une cryocapacitation, réduisant les chances de grossesse. L'hypotaurine est. naturellement présente dans les voies génitales masculines et féminines et possède des propriétés capacitantes, osmotiques et anti-oxydantes. Les mesures ont été réalisées sur le reliquat de sperme d'hommes avec des paramètres spermatiques normaux (n = 19) ou anormaux (n = 14). Les spermatozoïdes ont été sélectionnés par centrifugation sur gradient de densité (test de migration survie) avant congélation lente. Pour chaque prélèvement, ces étapes ont été réalisées en parallèle avec des milieux supplémentés en hypotaurine (bras « H+ ¼) ou sans hypotaurine (bras « H- ¼). Après décongélation, nous avons mesuré la mobilité totale et progressive, la vitalité, l'intégrité de l'acrosome, des marqueurs de la voie de signalisation de la capacitation et la qualité nucléaire. Pour cette dernière, nous nous sommes concentrés sur la condensation de la chromatine, la fragmentation de l'ADN et la présence de vacuoles dans le noyau du sperme. RéSULTATS: Post-décongélation, les spermatozoïdes sélectionnés et congelés en présence d'hypotaurine avaient une vitalité plus élevée (+ 16,7%, p < 0,001), une motilité progressive et totale (+ 39,9% et + 21,6% respectivement, p < 0,005) que les spermatozoïdes du bras « H- ¼ sans suplémentation. L'hypotaurine a également réduit la phosphorylation non spécifique des marqueurs protéiques de capacitation P110 et P80 (p < 0,01), indiquant une diminution de la cryocapacitation. La supplémentation en hypotaurine a réduit la décondensation de la chromatine, mesurée par la chromomycine A3 (− 16,1%, p < 0,05), la fragmentation de l'ADN (− 18,7%, p < 0,05) et la vacuolisation nucléaire (− 20,8%, p < 0,05). CONCLUSION: Notre étude est. la première à démontrer les effets bénéfiques de la supplémentation en hypotaurine dans les milieux de préparation et de congélation sur la capacité de fécondation des spermatozoïdes humains et leur qualité nucléaire. La supplémentation en hypotaurine a limité la cryocapacitation, augmenté la proportion de spermatozoïdes vivants et progressivement mobiles et réduit le pourcentage de spermatozoïdes présentant une décondensation de la chromatine, une fragmentation de l'ADN et une vacuolisation nucléaire. ENREGISTREMENT DE L'ESSAI: essai clinique, NCT04011813 . Enregistré le 19 mai 2019 - Enregistré rétrospectivement.

Sperm cryostorage in a dry tank: An accurate alternative.

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at -194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.

Comparison of human embryomorphokinetic parameters in sequential or global culture media.

A prospective study on randomized patients was conducted to determine how morphokinetic parameters are altered in embryos grown in sequential versus global culture media. Eleven morphokinetic parameters of 160 single embryos transferred were analyzed by time lapse imaging involving two University-affiliated in vitro fertilization (IVF) centers. We found that the fading of the two pronuclei occurred earlier in global (22.56±2.15 hpi) versus sequential media (23.63±2.71 hpi; p=0.0297). Likewise, the first cleavage started earlier at 24.52±2.33 hpi vs 25.76±2.95 hpi (p=0.0158). Also, the first cytokinesis was shorter in global medium, lasting 18±10.2 minutes in global versus 36±37.8 minutes in sequential culture medium (p <0.0001). We also observed a significant shortening in the duration of the 2-cell stage in sequential medium: 10.64 h±2.75 versus 11.66 h±1.11 in global medium (p=0.0225) which suggested a faster progression of the embryos through their first mitotic cell cycle. In conclusion, morphokinetic analysis of human embryos by Time lapse imaging reveals significant differences in five kinetic variables according to culture medium. Our study highlights the need to adapt morphokinetic analysis accordingly to the type of media used to best support human early embryo development.

Basonuclins and disco: Orthologous zinc finger proteins essential for development in vertebrates and arthropods.

Basonuclin 1 and the recently discovered basonuclin 2 are vertebrate proteins with multiple paired C(2)H(2) zinc fingers. It has long been known that the zinc fingers of basonuclin 1 closely resembled those of the Drosophila disconnected and discorelated proteins, two proteins essential for head development, but the relation between the basonuclins and the disco proteins has remained unclear because the putative function of basonuclin 1 in the control of keratinocyte growth potential appeared unrelated to that of disco and there was no resemblance between basonuclin 1 and Drosophila disco outside of the zinc fingers. The recent generation of a basonuclin-2 knockout has demonstrated that basonuclin 2 shares with disco a function in head development and the availability of new arthropod genome sequences has shown that the basonuclins are the vertebrate orthologs of the insect disco proteins. All these proteins are thought to be transcription factors, and it will have to be determined to what extent they share similar targets.

Basonuclin 2 has a function in the multiplication of embryonic craniofacial mesenchymal cells and is orthologous to disco proteins.

Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non-Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2.