Marcelle Cedars, M.D., Victor Fujimoto, M.D., Mitch Rosen, M.D., Heather Huddleston, M.D., Paolo Rinaudo, M.D., Anthony Dobson, M.D., and Shehua Shen, M.D. from the UCSF Department of Obstetrics and Gynecology and Reproductive Sciences are conducting a study to learn about ovarian stimulation and oocyte maturation to improve fertilization, embryo quality, implantation and clinical pregnancy rates in patients undergoing in vitro fertilization (IVF). Two hormones, follicle stimulating hormone and human chorionic gonadotropin (FSH/hCG) will be compared to the standard one hormone, hCG, for the ovulation trigger.

Over the past two decades, the success rate of assisted reproductive technology (ART) has dramatically increased. This increase has largely been attributed to improvements in the laboratory conditions and improvements in ovarian stimulation protocols (those medications used to increase the number of eggs maturing each cycle). Less work has been done on different ways to cause the final maturation of the eggs and the release of the egg from the ovary. The investigators propose to change the final injection prior to the egg retrieval (the ovulation trigger) so that it looks more like what happens in a normal menstrual cycle, where two hormones (both luteinizing hormone (LH) and FSH) increase. The investigators want to find out if this will improve egg quality and increase chances for pregnancy.

We observe during (In vitro fertilization) IVF, there are deviations in oocyte morphology and maturity within an individual cohort and that oocyte degeneration, and failed fertilization exist. Regardless, even if fertilization occurs, a large proportion of embryos fail to progress through the pre-implantation stages of development. In order for fertilization and embryo development to occur, the oocyte must mature or develop "competence". FSH may be a fundamental component to the final stages of oocyte maturation. Evidence suggests that with exogenous ovarian stimulation not all follicles achieve equal vascularity, and hence they are exposed to different amounts of FSH. We hypothesize FSH is required, within each follicle, at the time of ovulation trigger for oocyte maturation and prevention of atresia.

The ovarian stimulation prior to IVF attempts to mimic, and yet augment, normal physiology. The stimulation begins with gonadotropins to rescue antral follicles and stimulate growth. Subsequently hCG, which shares 80% homology with LH, is administered to facilitate maturation of the oocyte. However, the ovulatory phase in the normal menstrual cycle encompasses a concomitant LH and FSH prior to ovulation (see figure).

Maturation is a process whereby the oocyte undergoes changes in preparation for fertilization and embryo development. This entails both nuclear and cytoplasmic transformation. Nuclear maturation pertains to the resumption of meiosis to metaphase II (MII). It is well established that the LH surge is intimately involved in this process. Although the mechanism is not completely known, there are several steps. It is thought germinal vesicle breakdown requires a burst of calcium oscillations. During folliculogenesis, nuclear maturation of the oocyte is normally under tonic inhibition by a putative factor, oocyte-meiotic inhibitor (OMI). Some evidence suggests, prior to ovulation, LH inhibits the release of OMI from either the granulosa or theca cells. OMI likely acts as a paracrine factor and increases cAMP production in the granulosa cells (cumulus), which then acts as a messenger to the oocyte to maintain minimal calcium levels. In addition, LH is thought to decrease the gap junction communication between the cumulus and granulosa. Other evidence points to a putative signal that is synthesized by the granulosa cells, called follicular fluid meiotic activating substance. Both elements involved in nuclear maturation involve LH activity. The induction of LH receptors is via FSH. Under physiologic conditions there is a co-existent FSH surge with the LH surge. LH usually rises about 10 fold from baseline and FSH rises roughly 4 fold from baseline. It is possible that the surge of FSH ensures the required amount of LH receptors to complete nuclear maturation.

Cytoplasmic maturation is more difficult to identify. The process entails the synthesis of new proteins and post-translational modifications of existing proteins to allow for calcium activated pathways facilitating fertilization and embryo development. It is known that there is extensive cross-talk between the oocyte and granulosa cells. Few morphogenetic determinants of cytoplasmic maturation have been identified, but this remains an area of intense investigation From a clinical perspective, it is possible that FSH is required in this process of nuclear and/or cytoplasmic maturation, and that a minimal threshold of FSH may be required to maintain the gap junctions for completion of oocyte development. This evidence may be further supported by in-vitro maturation studies that show that FSH has a stimulatory effect on cytoplasmic and nuclear maturation.

Oocyte degeneration (atresia) is observed in 5-15% of the oocytes at the time of, or after, intracytoplasmic sperm injection. The etiology of degeneration has not been determined. The fate of the oocyte is likely determined prior to oocyte retrieval. At the time of retrieval, the apoptotic process in oocytes destined to undergo atresia has probably already been initiated. Under physiologic conditions, the granulosa cells die prior to the oocyte. There is evidence that atretic follicles have a high androgen to estrogen ratio. It is likely an indication of the deteriorating health of the granulosa cells. It is known that FSH has potent anti-apoptotic activity (inhibition of atresia), and the mechanism may be indirect via estradiol production. There is evidence that FSH primed follicles can grow with LH administration, in spite of low FSH levels. However, observations have shown that this process favors large follicles and that in the smaller follicles a critical ratio of FSH activity to LH activity is needed for survival. In support of this theory, others have suggested there is a narrow therapeutic window for LH. If E2 production is not adequate, LH may be detrimental to the follicle. The LH surge might hasten this process, in those follicles with a relative lack of vascularity (and/or lack of maturity), by a massive development of androgens and a relative lack of aromatase activity.

Inclusion Criteria:

• patients undergoing in vitro fertilization

Exclusion Criteria:

• risk of ovarian hyperstimulation syndrome

Adverse Event Data was collected between initial study visit (At Baseline) and final study visit (5 days after the oocyte retrieval procedure had been completed

Study adverse event and/or serious adverse event do not differ from the clinicaltrials.gov definition.

This study was powered to detect difference in fertilization instead of pregnancy outcomes, because the number of patients required to detect a 10% absolute difference in pregnancy rates was too large to be completed at our center (n=387 per group).