155 LAPAROSCOPIC EVALUATION OF OVARIAN REACTION TO HORMONE STIMULATION IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)

Abstract

Oocyte collection is a key step to development of a system for modeling human regenerative medicine and assisted reproductive technology in cynomolgus monkeys (Macaca fascicularis), but collecting oocytes at a suitable developmental stage (metaphase II) is difficult. Metaphase II (MII) oocytes can be collected from cynomolgus ovaries stimulated by hormone injection. In this study, we developed a useful method for collecting a large number of MII oocytes by monitoring the morphology and size of ovaries with laparoscopic observation. Controlled ovarian stimulation and oocyte recovery in mature cynomolgus monkeys have been previously described by Torii et al. (2000 Biochemistry 39, 3197–3205). Beginning at menses, levels of estrogens were down-regulated by the subcutaneous injection of a GnRH antagonist (Leuplin, 0.9 mg animal^-1; Takeda Chemical Industries, Ltd., Osaka, Japan). Two weeks later, human follicle stimulating hormone (FSH, Fertinorm, 25 IU kg^-1; Serono, Canton Zug, Switzerland) was administered for 9 days. On the day after that of the last FSH administration, human chorionic gonadotropin (hCG, Puberogen, 400 IU kg^-1; Sankyo Co., Ltd., Tokyo, Japan) was intramuscularly injected. Follicular aspiration was performed at 404-41 hours post-hCG injection. Oocyte collection was monitored using a 3-mm laparoscope attached to a video system. Oocytes were aspirated from the follicles using a 20-guage needle. Follicle development of the ovary was rated morphologically as A (small follicles), B (many small follicles), or C (many large follicles), and size relative to the uterus was rated as 1 (no response), 2 (smaller), 3 (equal), or 4 (larger) at oocyte collection. Regarding morphology, the highest ratio of MII/total oocytes was obtained from B–C ovaries (rating of 2 ovaries, one each left or right with a rating of B or C, n = 8, 26.9 ± 22.4, 70.7%), followed by B–B (n = 21, 19.5 ± 14.3, 55.9%), and C–C (n = 19, 10.6 ± 5.8, 51.9%). No MII oocytes were collected from A–A (n = 1, 0%) ovaries. Ovaries appeared to be over-stimulated in the ovaries rated C–C but under-stimulated in B–B. Regarding ovary size, the highest ratio of MII/total oocytes was obtained from 4–4 (both ovaries with rating of 4, n = 10, 21.9 ± 11.2, 68.8%), followed by 3–3 (n = 21, 19.1 ± 14.3, 58.2%), and 2–2 (n = 9, 11.6 ± 6.5, 53.3%). No MII oocytes were collected from 1–1 ovaries (n = 1, 0%). The number of MII oocytes collected was directly related to ovary size: more MII oocytes were collected from larger ovaries. These data demonstrate that the number of oocytes collected is directly related to ovary size. Our results suggest that the ratio of MII oocytes can be predicted by the morphology and the size of ovaries. In addition, we found that ovarian development can be controlled by adjusting FSH dosage. Therefore, laparoscopic observation of ovaries during FSH treatment and adjusting FSH dosage are necessary to collect MII oocytes efficiently.