Human tubal fluid medium

Controlled ovarian stimulation was performed mainly by the agonist/human menopausal gonadotropin (hMG) method, antagonist/hMG method, and clomiphene citrate/antagonist/hMG method. Oocyte retrieval was performed under the guidance of transvaginal ultrasound 36–38 h after the trigger by hCG or GnRHa agonist.
While examining oocytes, cumulus‐oocyte complexes were stretched, and the presence or absence of the first polar body was confirmed using an inverted microscope (IX70, Olympus). The oocytes were divided into MI oocytes and MII oocytes. Among the MII oocytes, the oocytes with fragmented polar bodies that appeared to have divided into two parts were separated and cultured. After oocyte retrieval, a maximum of five oocytes were added to the Center Well Dish containing 1 ml of human tubal fluid medium (Irvine Scientific, Santa Ana, USA) and then cultured.
For IVF, insemination was performed at least 3 h after preculture following oocyte retrieval. Standard insemination sperm concentration was set at 200 000/ml and corrected based on normal sperm morphology ratio and previous fertilization rate. ICSI was selected for cases where the adjusted motile sperm count was insufficient for the sperm count for insemination, as well as for cases in which TFF had occurred in a previous round of IVF. ICSI was performed at least 3 h after preculture.

Cyclophosphamide- or saline-injected mice were superovulated via intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (Prospec, Rehovot, Israel), followed by 5 IU human chorionic gonadotropin (hCG, Prospec) at 48 h later. Oocytes were collected 18 h post-hCG injection in preincubated Human Tubal Fluid medium (Irvine scientific, CA, USA). Oocytes were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) for 10 min. Oocytes were blocked in phosphate-buffered saline containing 3% bovine serum albumin (Genedepot, Katy, TX, USA), and then incubated with rabbit anti-α-tubulin antibody (1:200, Cell Signaling Technologies, Danvers, MA, USA). Oocytes were mounted with VECTASHIELD Antifade Mounting Medium with 4′6’-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) to visualize the chromosomes, and observed by fluorescence microscopy (Olympus BX51, Tokyo, Japan). Oocytes with well-organized, bipolar spindles and chromosomes that were tightly aligned at the metaphase plate were scored as normal. Oocyte quality was also evaluated by measuring morphometrical parameters, including the complete oocyte, ooplasm, and perivitelline space (PVS) using NIS-elements BR 4.60.00 software (Nikon, Tokyo, Japan) [16 (link)].