Three-dimensional in vitro maturation of rabbit oocytes enriched with sheep decellularized greater omentum.

BACKGROUND: To prevent ovarian hyperstimulation syndrome, in vitro maturation (IVM) allows the oocytes for infertility treatment without hormone therapy. Although many oocytes matured during IVM, some deficiencies in the culture conditions lead to inhibition of the growth and development of the cumulus cells and the oocyte nuclear and cytoplasmic maturation. OBJECTIVES: The challenge of improving the oocyte culture conditions prompted us to use greater omentum (GOM), full of growth factors and proteins, as a rich supplement to the base culture medium. METHODS: Cumulus-oocyte complexes were recovered from rabbits and divided into 3D and 2D conditions cultured for 12 and 24 h. In 3D cultures, the oocytes embedded in alginate containing FBS decellularized GOM. Corresponding supplements were also added in 2D conditions-maturation of the oocytes evaluated by Aceto-Orcein, TEM, and RT-PCR for MAP2K1 and Cdk2. RESULTS: DNA quantification, Hoechst, and H&E staining confirmed cell depletion from GOM, and SEM showed the preservation of ultra-architecture after decellularization. Histochemical staining methods showed appropriate extracellular matrix preservation. ELISA assessment showed retention of VEGF content. MTT assessment indicated decellularized GOM was non-toxic. Both Aceto-Orcein assessment and ultra-structure study of the oocytes showed that supplementation of 2D or 3D cultures with decellularized omentum promoted oocyte maturation. Expression of MAP2K1 and Cdk2 also increased in the presence of GOM. CONCLUSIONS: GOM supplementation has a beneficial impact on oocyte maturation, probably due to the presence of growth factors and proteins.

Lectin reactivity of expanded mouse blastocysts after exposure to sera from women with unexplained recurrent spontaneous abortion.

Embryotoxic factors existing in maternal sera may influence their effects via specific binding to, or alteration of cell surface molecules in the conceptus. This study was undertaken to determine the effects of sera from women with unexplained recurrent spontaneous abortion (URSA) on cell surface glycoconjugates of the early conceptus. Four cell stage embryos were cultured in medium supplemented with sera from women with URSA, from normal women, or in medium without serum. Developmental competence was assessed as the stage distribution of embryos advancing to during 96h in culture. Hatched (expanded) blastocysts were stained with wheat germ agglutinin (WGA), peanut agglutinin (PNA) and dolichos biflorus agglutinin (DBA) to detect surface glucoconjugates. We observed that patient sera could be divided into high- and low-risk groups on the basis of the ability to decrease the number of four-cell embryos reaching the expanded blastocyst stage. Furthermore, the intensity of reactivity to PNA changed after exposure to high-risk sera. Morula formation was reduced and blastocyst formation was delayed. Although the sera from women with URSA had embryotoxic effects, no influence on the glycoconjugate patterns were evident in hatched blastocysts, aside from PNA reactivivity. We suggest altered developmental display of PNA-reactive proteins was a biomarker for poor developmental quality due to emrbyotoxic factors in serum from URSA patients.