Use of a novel long-acting human follicle-stimulating hormone analogue containing four extra n-linked carbohydrates in the ovarian hyperstimulation of a female baboon

Abstract

Objective: A single chain human follicle-stimulating hormone (FSH) analogue with four additional N-linked glycosylation sites (rhFSH-N4) has recently been shown to prolong elimination half-life (T1/2) and increase biopotency as compared to commercially available recombinant human FSH (rhFSH) in the murine model. Longer-acting gonadotropins may improve patient compliance and comfort by requiring fewer injections in women undergoing controlled ovarian hyperstimulation (COH) protocols for in vitro fertilization (IVF). Superovulation with rhFSH given daily and collection of oocytes via aspiration has been examined in the baboon. Use of rhFSH-N4 in baboons for the purpose of COH and collection of multiple oocytes via uterine lavage, as well as T1/2 determination of the analogue, were examined. Design: Experimental study. Materials and Methods: A multiparous, regularly cycling female baboon was chosen to undergo COH using experimental rhFSH-N4. Ovarian stimulation was initiated on day 1 of the menstrual cycle with a subcutaneous (SC) injection of rhFSH-N4 (75 IU). A 2nd dose of the rhFSH-N4 (75 IU) was given on day 5 when serum estradiol (E2) levels had plateaued. A GnRH antagonist (250μg SC) was given daily starting on day 6 until the day of hCG administration. Serum E2, FSH, and progesterone levels were measured daily. Pelvic ultrasound was performed under anesthesia (ketamine/xylazine) every other day to monitor number and size of follicles until hCG was given. Recombinant hCG (250μg SC) was given when majority of follicles were >5mm in diameter. 7 days post-ovulation, uterus was lavaged under anesthesia with 10ml of warm, sterile, isotonic, culture medium using a 27-g double-barreled cannula through the cervical canal to collect unfertilized oocytes. Results: Serum E2 levels started to rise on day 3 and plateaued after 5 days, requiring a 2nd dose of rhFSH-N4 to be given. Serum E2 peaked and plateaued by day 6–9 of stimulation. Follicular development was not observed until day 8–9. Majority of the follicles (n=7) increased to >5mm in diameter on day 10 when hCG was given. No oocytes were collected via uterine lavage 7 days post-ovulation. Serum FSH levels increased after each dose of rhFSH-N4, and its T1/2 was 48 hours. Serum progesterone levels followed a pattern consistent with ovulation after administration of hCG. Conclusions: Prolonged T1/2 of rhFSH-N4 in a primate species was confirmed. Use of rhFSH-N4 in baboons as part of a modified human COH protocol is successful in inducing follicular development as evident by increased E2 production and growing follicular size, while requiring fewer injections as compared to commercially available rhFSH. However, no oocytes were collected via uterine lavage. Protocol needs to be modified to account for the delayed tubal transport that may accompany hyperstimulation. Tubal lavage, although more invasive, may be considered to increase yield of oocyte collection if inadequate results persist despite delaying the time of uterine lavage. Once the problem of oocyte collection is cleared, the protocol can be modified to allow animals to be bred during the COH cycle with the goal of collecting multiple embryos to isolate baboon embryonic stem (ES) cells, which may provide an in vitro model of ES cell differentiation similar to human ES cells. Ultimately, use of a long-acting hFSH analogue for COH in women undergoing IVF may be beneficial by requiring fewer injections, thereby increasing comfort and compliance.