339 CORRELATION BETWEEN FOLLICLE SIZE AND QUALITY OF OOCYTES FROM THE SUPEROVULATED CYNOMOLGUS MONKEY

Abstract

Variability in the superovulatory response continues to be one of the most frustrating problems with the application of assisted reproductive technologies in non-human primates. Superstimulation of donor animals with equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) is widely used, but individual responses to these hormones vary widely. In human in vitro fertilization, follicle size is commonly used as a marker to determine the timing of hCG treatment in order to acquire oocytes matured in vivo. Incorporation of techniques designed to control follicular size in humans may improve superstimulatory response in non-human primates. In this study, we measured follicle size and examined oocyte quality in Cynomologus Monkeys superstimulated as described below. On the first day of spontaneous menses, monkeys were treated with long-acting GnRH (Luprin: Takeda Pharm, Co. Ltd., Osaka, Japan;; Day 0). A dose of 25 IU/kg/day eCG (Serotoropin;; Teikokuzoki Pharm, Co. Ltd., Tokyo, Japan) was administered i.m. from Day 1 to Day 9, followed by 200 IU kg⁻¹ hCG (Puberogen;; Sankyo Pharm, Co. Ltd., Tokyo, Japan) i.m. on Day 10. Dominant follicle sizes were measured on Days 7 and 9 by ultrasonography. Oocytes were collected by laparoscopy in anesthetized monkeys 40 h after the hCG injection. Oocytes were classified by nuclear status. Immature oocytes at the stages of germinal vesicles (GV) and metaphase (MI) were cultured until reaching the stage of Metaphase II (MII). Matured oocytes (MII) were fertilized by ICSI and cultured for 7 days. At the end of culture, the developmental stage of oocytes was examined. The ovaries with different-sized follicles on Day 7 were divided into two groups;; ovaries with large follicles (>4.5 mm) were in the first group and ovaries with small follicles (<4.0 mm) were in the second group. On Day 9, follicles in first group grew to more than 5.0 mm and follicles in second group remained less than 5.0 mm. Sixty-two percent of oocytes from follicles in first group were at MI or MII stage, while only 15% of oocytes in second group reached the MI or MII stage. After ICSI, 42% of MII oocytes from first group developed to the blastocyst stage, while no blastocyts were observed in second group. These results suggest that the size of dominant follicle was a limiting factor for the developmental ability of oocytes in vitro. For production of Cynomolgus monkey blastocysts derived from ICSI, the diameter of dominant follicle was required to be at least 5 mm before hCG in order to collect MI and MII oocytes. Incorporation of hormonal treatments designed to optimize follicular size probably reduced the variability in quality of oocytes. Therefore, we expect that an adjustment of dose and duration of eCG and hCG treatment may improve developmental ability of oocytes from follicles that had not reached 5 mm.