Calcium ionophore supplementation for immature oocytes and pentoxifyllin supplementation for immotile sperms : A study of embryo quality of difficult cases in in vitro fertilization_Data
Description
This data describes the oocyte maturation of immature oocyte caused of Ca-I and motility increase of immotile sperm caused of PTX supplementation.
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Male mice were divided into 3 groups, namely native/pre-washed necrozoospermia, post-washed necrozoospermia with and without pentoxyphilin (PTX). Same as male, the female mice were also devided into 3 groups, namely mature oocytes, immature oocytes with and without calcium ionophore (CaI). Oocyte source and maturation The mice were stimulated using recombinant FSH (Gonal F) at a dose of 5 IU twice with a break between injections of 48 hours. (Gonal-F, Merck, Denmark), while ovulation was triggered using 10 IU of the LH analog (Ovidrel, Merck, Denmark) injected intraperitoneally. The denuding process was performed using hyaluronidase enzyme. The immature oocytes were stored in Synvitro medium Flush to which calcium ionophore A23187 (CaI) has been added with a concentration of 5 µM, then incubated for 5 minutes (Handayani et al., 2020; Nikiforaki et al., 2016). The oocytes are then washed and cultured in universal IVF medium for 20 - 24 hours, then assessed whether they are mature or not. Sperm Collection, Preparation and PTX supplementation After euthanasia, sperm obtained from the cauda epididymis by making a notch in the cauda. Native groups were treated with 200 µL of sperm rinse solution (Vitrolife Cat. No. 10101, Sweden) and centrifuged at 1800 xg for 10 minutes. The pellet resuspended in 100 µL of EmbryoMax® Human Tubal Fluid (HTF) medium (Sigma-Aldrich Cat No.MR-070-D). In the treatment group, pentoxifylline was added at a dose of 10 µM after preparation for 30 minutes. Sperm can then be used for IVF (Khalili et al., 2017; Kovačič et al., 2006). Intracytoplasmic sperm injection (ICSI) and embryo development The oocyte was placed in a GMOPS drops (Vitrolife, Sweden) which was covered by liquid paraffin. At ICSI, a chosen sperm was then dragged using an injection pipette. Then, the injected oocyte was transported to the G1 medium (Vitrolife, Sweden) to be keep warm inside a triple-gas incubator (5% O2, 6% CO2, and 5% N2) with a temperature of 37C. After day-2 had entered the 6-8 cell stage, the embryo would be transported to the G2 medium (Vitrolife, Sweden). The modification of Gardner system was used to observe embryo development quality. Statistical analysis Oocyte maturity data with/without calcium addition is nominal data so it will be analyzed using the chi square test. Data on spermatozoa motility with/without the addition of pentoxifylline is numerical data and will be analyzed using the Kruskal Wallis test, followed by the Mann-Whitney post hoc test. Comparison of embryo quality data was tested using the Kruskal Wallis test.
Institutions
- Universitas Indonesia