In Vitro Fertilization and Embryo Transfer in the Rhesus Monkey1

Abstract

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.

Chromosomal instability in rhesus macaque preimplantation embryos

“…Procedures for oocyte recovery, sperm collection, insemination and embryo culture have been described previously (22)(23)(24). Briefly, follicular fluid aspirates were collected in TALP-Hepes containing 0.3% bovine serum albumin (BSA).…”

In Vitro Manipulation of Nonhuman Primate Gametes for Embryo Production and Embryo Transfer.

“…An IVF technique has been developed and successfully used for several types of nonhuman primates [20,21,24,27,38,40,42,72,80,112,114,115,117]. For example, we first reported IVF with frozen-thawed spermatozoa from cynomolgus monkeys, in which the embryo obtained developed to the hatched blastocyst stage [84] (Fig.…”

“…Our research strategy was the first to choose sexually mature females with normal menstrual cycles, and then to select from these candidates those females whose cycles were in synchronization with the embryo stage. When cryopreserved embryos are used, this synchronization is relatively simple to control: the embryos are thawed so as to be in synchronization with the menstrual cycle of the recipient, and then they are transferred [4,54,75,115,119].…”

In vitro fertilization‐embryo transfer in nonhuman primates: The technique and its applications

“…The latter may occur in immature follicles due to excessive exogenous LH or in ovulatory follicles due to a spontaneous LH surge. We currently measure circulating levels of estradiol and progesterone in hyperstimulated rhesus monkeys Wolf et al, 1989). If estrogen levels decline (> 20% of the previous daily value) or progesterone concentrations rise (> 1 ng/ml before hCG treatment), circulating levels of bioactive LH are measured in retrospect to determine the incidence of a n LH surge .…”