Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg′ of freeze-drying buffer and temperature during the drying phase.ArticleCRYOBIOLOGY. 68(3):354-360 (2014)journal articl

Genotoxic risk and oocytes : mouse oocytes Genotoxicity assessment of cryoprotectant and oocyte vitrification protocols.

La toxicologie génétique est une discipline qui vise à détecter des facteurs chimiques ou physiques interagissant avec l'ADN des cellules et qui, en l'absence de réparation fidèle, sont susceptibles de provoquer des mutations géniques et/ou chromosomiques. Le test des comètes est un test court de génotoxicité simple, reproductible et rapide pour étudier la survenue de lésions primaires de l'ADN. Il s'agit d'une technique micro-électrophorétique très sensible permettant la mise en évidence des lésions de l'ADN de cellules eucaryotes individuelles exposées à des agents génotoxiques. En présence de cassures de l'ADN, les fragments d'ADN ainsi formés migrent plus rapidement que l'ADN intact lors de l'électrophorèse, donnant aux noyaux cellulaires l'aspect de comètes. La cryoconservation des ovocytes matures par vitrification a de nombreuses applications: alternative à la congélation d'embryons en FIV, préservation de la fertilité avant traitement gonadotoxique, développement du don d'ovocyte. La vitrification consiste à transformer un liquide en un état vitreux et utilise des agents cryoprotecteurs (CP) à haute concentration. Plusieurs centaines de naissances d'enfants en bonne santé ont été décrites . Néanmoins peu d'études ce sont intéressées aux effets à long terme de cette technique en particulier au plan génétique. L'objectif de ce travail a été dans un premier temps de développer et de valider une technique de test des comètes sur ovocyte de souris, puis d'utiliser ce test pour évaluer la génotoxicité du PrOH sur les ovocytes de souris qu'il soit employé seul ou inclus dans les solutions de vitrifications commercialisées pour la cryoconservation d'ovocytes humains.Genetic toxicology is a discipline that aims to detect chemical or physical factors interact with the DNA of somatic and / or germ cells and in the absence of accurate repair, are likely to cause gene and / or chromosomal mutations. The comet assay is a simple, reproductive and rapid test to study primary DNA damage. This microelectrophoretic technique, is used to visualize denatured DNA fragments migrating out of the cell nucleus during electrophoresis. The image obtained is a ''comet'' with a distinct head consisting of intact DNA and a tail containing relaxed DNA loops or broken pieces of DNA. Oocyte vitrification techniques is booming in the world, with the key to many applications: alternative to IVF embryo freezing, fertility preservation before gonadotoxic treatment, development of oocyte donation. Oocyte Vitrification traps all the aqueous solutions in a vitreous solid phase, preventing any ice crystal formation, because of very high cooling rates and high cryoprotectants concentrations. vitrification-cryopreservation has led to several hundred live births with reassuring obstetrical and perinatal outcomes. However, little is known about the possible long-term consequences on human live births after oocyte vitrification. The objective of this work was initially to develop and validate a technique comet assay on mouse oocyte, then to evaluate on mature oocytes the genotoxic effects of PrOH solution and finally the genotoxic effects of three oocyte vitrification protocols used in human ART the genotoxic of three oocyte vitrification protocols used in human

Appropriateness of standardised enhanced recovery program following caesarean section. Survey and improvement of professional practices

International audienceno abstrac

Étude pronostique du risque d’accouchement, des patientes hospitalisées pour menace d’accouchement prématuré, dans une maternité de niveau III

International audienceno abstrac

Ultrastructural Interactions and Genotoxicity Assay of Cerium Dioxide Nanoparticles on Mouse Oocytes

International audienceCerium dioxide nanoparticles (CeO2 ENPs) are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with CeO2 ENPs to study (1) physicochemical biotransformation of ENPs in culture medium; (2) ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM); (3) genotoxicity of CeO2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of CeO2 ENP aggregates in follicular cells. In oocytes, CeO2 ENP aggregates were only observed around the zona pellucida (ZP). The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of CeO2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP