Intracytoplasmic injection of frozen-thawed epididymal spermatozoa in a nonhuman primate model, the cynomolgus monkey ( Macaca fascicularis)

Abstract

Intracytoplasmic sperm injection (ICSI) with frozen-thawed epididymal spermatozoa was performed in the cynomolgus monkey ( Macaca fascicularis) to produce embryos in vitro. Eleven sexually mature females were hyperstimulated with an GnRH agonist (1.8 mg active triptorelin per 2 kg body weight), followed (2 weeks later) by rFSH (37.5 IU per 2 kg daily) for 12 days, and finally 1000 IU of hCG. Epididymal spermatozoa were collected from a single adult male monkey. The first stimulation cycle resulted in 90 oocytes; 70% of which were metaphase II (MII). Sixty-four percent of these MII oocytes were fertilized. Comparing ovarian response of five monkeys that underwent a second stimulation cycle there was an increase in oocyte quantity (13.2 versus 9.2 oocytes per monkey) but the percentage of MII oocytes remained the same at 58%. Fertilization and cleavage rates were also reduced but there was an increase in the number of embryos available for transfer. Overall, four monkeys became pregnant resulting in the birth of two healthy infants and two abortions. These findings show that [1] ovarian stimulation by GnRH–rFSH did not compromise the developmental competence of the oocytes; [2] effective combination of cryopreservation of epididymal spermatozoa and ICSI is possible in nonhuman primate reproduction, and thus has potential application in the conservation of highly endangered nonhuman primate species, and [3] the cynomolgus monkey is a reliable biomedical research model to study the potential risks and benefits associated with assisted reproductive techniques prior to approval for clinical trials on humans.