Overriding follicle selection in controlled ovarian stimulation protocols: quality vs quantity

Abstract

Selection of the species-specific number of follicles that will develop and ovulate during the ovarian cycle can be overridden by increasing the levels of pituitary gonadotropin hormones, FSH and LH. During controlled ovarian stimulation (COS) in nonhuman primates for assisted reproductive technology (ART) protocols, the method of choice (but not the only method) has been the administration of exogenous gonadotropins, either of nonprimate or primate origin. Due to species-specificity of the primate LH (but not FSH) receptor, COS with nonprimate (e.g., PMSG) hormones can be attributed to their FSH activity. Elevated levels of FSH alone will produce large antral follicles containing oocytes capable of fertilization in vitro (IVF). However, there is evidence that LH, probably in lesser amounts, increases the rate of follicular development, reduces heterogeneity of the antral follicle pool, and improves the viability and rate of pre-implantation development of IVF-produced embryos. Since an endogenous LH surge typically does not occur during COS cycles (especially when a GnRH antagonist is added), a large dose of an LH-like hormone (i.e., hCG) may be given to reinitiate meiosis and produce fertilizable oocytes. Alternate approaches using exogenous LH (or FSH), or GnRH agonist to induce an endogenous LH surge, have received lesser attention. Current protocols will routinely yield dozens of large follicles with fertilizable eggs. However, limitations include non/poor-responding animals, heterogeneity of follicles (and presumably oocytes) and subsequent short luteal phases (limiting embryo transfer in COS cycles). However, the most serious limitation to further improvements and expanded use of COS protocols for ART is the lack of availability of nonhuman primate gonadotropins. Human, and even more so, nonprimate gonadotropins are antigenic in monkeys, which limits the number of COS cycles to as few as 1 (PMSG) or 3 (recombinant hCG) protocols in macaques. Production and access to sufficient supplies of nonhuman primate FSH, LH and CG would overcome this major hurdle.

Figure 1

Diagram of the events occurring in the ovary and reproductive tract during the initial three weeks of the fertile menstrual cycle leading to natural reproduction in primates.

Figure 2

Diagram of events occurring in the ovary and in vitro during controlled ovarian stimulation cycles leading to assisted reproduction in primates. This chapter will discuss the methods and their limitations for increasing circulating levels of gonadotropins (FSH, LH, CG) to override the typical selection and maturation of a single "dominant" follicle in the natural menstrual cycle, thereby stimulating the development and maturation of multiple large follicles whose oocytes can be collected for in vitro manipulation (e.g., in vitro fertilization, IVF) prior to return to the reproductive tract (embryo transfer, ET) for pregnancy initiation.

Figure 3

Histologic sections of ovaries. Ovaries were removed from rhesus monkeys after 90 days of treatment with GnRH antagonist prior to (left panel) and following administration of r-hFSH and r-hLH (right panel). Note the absence of any large (>1 mm diameter) antral follicles following GnRH antagonist exposure, versus the development of 2–6 mm antral follicles after 9 days of gonadotropin treatment. See text, and ref [39] for further details.

Figure 4

Illustration of the heterogeneity in follicle size following controlled ovarian stimulation in rhesus macaques. The number and size of antral follicles that develop on the ovaries after daily treatment with exogenous gonadotropins can be estimated by transabdominal ultrasonography (left panel). The percent of the total follicle cohort at various sizes ≥ 2 mm diameter on day 7 of our standard COS protocol (6 days of r-hFSH, 30 IU 2× per day, then r-hFSH + r-hLH, 30 IU each 2× per day; plus daily GnRH antagonist treatment [15]) are illustrated in the right panel. Typically, in this and prior [39] protocols, follicles vary in size between 2 to 6–8 mm in diameter.

Figure 5

Morphology (left panel) and percent (right panel) of atretic follicles. Follicles were exhibiting histologic evidence of atresia (pyknotic, unadhered granulosa cells in the antrum; left panel) following administration of the hCG bolus in COS protocols in rhesus monkeys. Although relatively few (<10%) appear atretic prior to hCG injection (0 hr; small number below bar indicates sample size), the number increases significantly by 36 hrs post-hCG (40%; controls, CTRL). Moreover, the percentage of atretic follicles was influenced by the steroid milieu, since administration of a steroid synthesis inhibitor (trilostane, TRL) produced a cohort of 70% atretic follicles and co-administration of a progestin (R5020) reduced the percentage to pretreatment (0 hr hCG) levels of 10%. Thus, oocytes collected prior to follicle rupture could originate from degenerating, as well as healthy (i.e., luteinizing, ovulatory) follicles. See ref [59] for further details, including statistical (X²) analyses. Different letters above bars indicate significant differences over time within controls (CTRL; hCG alone). Asterisk, or NS indicate significant or nonsignificant differences between treatment groups at one timepoint.