Widjiati

Effect of Insulin Transferrin Selenium Administration on Rat's Cultured In Vitro Embryo Post Warming After being Frozen using Vitrification Method

Success of embryo transfer is determined by availability of a plenty of amount of embryo stock. To make embryo stock remain in good quality, it is necessary to have an easy, cheap, simple and effective storage method. One of the storage methods is vitrification.Success of vitrification method is still disrupted due to decreased embryo quality post warming. Decreased embryo viability influences high implantation rate and gestation. Low implantation rate will result in low gestation rate. Therefore, study is needed to optimize vitrification medium so that it will be able to optimize cryoprotectant role to protect embryo due to temperature stressor in vitrification method.Administering Insulin Transferrin Selenium on vitrification medium is able to bind free radicals caused by temperature stressor due to frozen Insulin Transferrin Selenium which is a complex protein that is able to stimulate cell growth, prevent cell damage due to anti oxidant role in it so that it is able to maintain embryo viability post thowing. Insulin Transferrin Selenium is able to increase quality and viability of blastocyst resulted from in vitro culture.The study aims to prove Insulin Transferrin Selenium supplementation on vitrification medium is able to increase viability of rat embryo post warming. Steps of the research cover oocyte collection, embryo vitrification, warming, and in vitro culture. In vitro culture yields morula embryo with excellent quality and fits to be frozen using vitrification method.Insulin Transferin Selenium administration of 5,10 and 15 g/ml is able to increase embryo viability up to 100 %. Frozen embryo post warming and recultured for 5 hours , viability of morula embryo ofthe treatment group administered by Insulin Transferrin Selenium reached 73.4 % 83.4% whereas control group without being administered by InsulinTransferrin Selenium reached only 64.7 %. The research concludes that Insulin Transferrin Selenium.administration is able to increase viability of rat embryo at morula stage.

Hyaluronan Expression on Vitrified Oocytes Before and After In Vitro Maturation (EKSPRESI HYALURONAN PADA OOSIT YANG DIVITRIFIKASI SEBELUM DAN SESUDAH MATURASI IN VITRO)

Oocyte vitrification is a major challenge in assisted reproductive technology. Oocyte vitrification with cumulus cells provide benefits in the process of maturation and fertilization. Vitrification leads to rapid temperature changes, therefore the decreasing in temperature could damage the cells even when the morphology was normal. Vitrification of mature oocytes is common because of its low sensitiveness towards low temperatures than immature oocytes. The aim of the research was to compare the effect of vitrification before and after in vitro maturation to the expression of hyaluronan. Maturation was operated in medium TC 50 μL in CO 2 incubators for 24 hours. Vitrification started with washing oocyte in PBS basic medium supplemented with 20% serum for 1-2 minutes, then in equilibration medium PBS + 20% serum + 10% ethylene glycol for 10-14 minutes, then transferred to 20% serum + PBS + 0.5 M sucrose + 15% ethylene glycol + PROH 15% for 25-30 seconds. Thawing was processed by submerging the oocytes in the media: 1). PBS + 20% serum + 0.5 M sucrose (K1); 2) PBS + 20% serum + 0.25 M sucrose (K2); and 3).PBS + 20% serum + 0.1 M sucrose (K3). Immunocytochemical stain was performed to evaluate the hyaluronan expression. Remmele scale index (Immunoreactive score, IRS) was used to read the result. There was no differences of hyaluronan expression in oocyte and cumulus group of K1, K2 and K3 at p< 0.05, statistically. We concluded that there was no difference of hyaluronan expression on oocyte and cumulus between vitrified oocyte of pre and post in vitro maturation which indicated that oocyte could be vitrified in the immature and mature state.

Effectiveness of Insulin Transferrin Selenium Supplementation to Vitrified Mice Using Hemi Straw on Zona Hardening : Expression of Heat Shock Protein 70 and Caspase 3

Addition of Insulin Transferrin Selenium in vitrification medium can scavenge free radicals caused by temperatures stressors due to the freezing. Insulin Transferrin Selenium is complex protein that can stimulate cell growth, prevent cell damage due to the role of antioxidants inside, so it can maintain the viability of the embryo after thawing. Insulin Transferrin Selenium can improve the quality and viability of in-vitro blastocyst culture results. The purpose of this study is to prove the effectiveness Insulin Transferrin Selenium supplementation in vitrification medium can reduce the hardening zone of mouse embrio post thawing, proving the effectiveness of Insulin Transferrin Selenium supplementation in vitrification medium on the expression of Heat Shock Protein 70 in mouse embryos post thawing, and prove the effectiveness of Insulin Transferrin Selenium supplementation the vitrification medium can decrease the expression of caspase 3 in mouse embryos post thawing. This study covered the stages of superovulation and collection eggs, in-vitro fertilization (IVF), modification vitrification medium with supplementation of Insulin Transferrin Selenium (ITS), embryo vitrification with hemi straw, embryo culture, examination of the hardening zone of mouse embryo, examination of Heat Shock Protein 70 expression and examination of caspase 3 expression in embryos post thawing.The results showed that there were differences between the addition or supplementation of Insulin Transferrin Selenium and without addition of Insulin Transferrin Selenium in the vitrification medium to hardening zone and caspase 3, but it was no differentiation in the expression of Heat Shock Protein70. The conclusion is the supplementation of Insulin Transferrin Selenium in vitrification medium decreases the hardening zone of the mouse embryos post thawing, decreases the caspase3 expression in the mouse embryos post thawing and there is no difference in the expression of Heat Shock Protein70 in the mouse embryos post thawing