Can Dormant Perimenopausal Ovarian Follicles Become FSH Responsive?

Introduction Women stop menstruating at the mean age of 51, yet pregnancies become much less prevalent beyond 40, with pregnancy at 43 being a relative rarity. Current common perception attributes the regression of fertility to depletion of the primordial follicles' pool (Burger et al. 2008). The decline in fertility may result not necessarily from a sharp depletion of the primordial follicle pool (Hansen et al. 2008) but may be due to failure of adequate transition from primordial to antral, gonadotropin-responsive follicles. It is not infrequent to encounter a fertility patient at her late thirties or early forties, who would not respond to the most extreme doses of exogenous FSH, but will nevertheless continue to menstruate until the age of 50. The usual practice is to refer these patients to oocyte donation programs.

Recently, Li et al. (2010) demonstrated that incubation of neonatal mouse ovarian fragments with an inhibitor of the Phosphatase with TENsin homology deleted in chromosome 10 (PTEN) phosphatase and a PI3K activating peptide, increased nuclear exclusion of Foxo3 and propelled large numbers of dormant primordial follicles into becoming FSH-responsive antral follicles. Furthermore, in the nude mice model (mice lacking immune system) they showed that transplanted human ovarian fragments containing mainly primordial follicles from surgical specimens, treated by the same incubation, also underwent activation of dormant follicles to derive preovulatory follicles containing mature oocytes.

Based on all the above we hypothesize, that ovaries of women at their perimenopausal years could be propelled to become FSH-responsive, using incubation with PTEN inhibitor and PI3K activating peptide to activate dormant primordial follicles.

If found feasible, this technique could provide hope of achieving fertility to patients who would otherwise be referred to receive oocyte donation.

Aim of the proposed study To obtain ovarian cortical fragments from perimenopausal patients, who undergo gynecological surgery, and test whether treatment with PTEN phosphatase and PI3K activating peptide would induce these fragments to produce FSH responsive follicles in the nude mouse model.

Study subjects and specimen harvest Up to ten women, older than 45 years, who are scheduled to gynecological operation, will be asked to provide half of an ovary for the study. The tissue will then be cut to multiple fragments, suitable for cryopreservation.

Laboratory methods Cryopreservation of ovarian fragments will use a vitrification protocol similar to earlier report for oocyte cryo-storage (Yoon et al. 2003). Because the cryo-preservation reagents can easily penetrate primordial follicles as compared with the large oocytes, we anticipate efficient preservation of these follicles. In young patients, one 1 mm cortical cubes contains ~50 primordial follicles.

Three tissue samples from each patient will be transferred to the Hsueh laboratory in Stanford (attached letter), and will be thawed for in vitro activation followed by xeno-transplantation using the same protocol as that described by Li et al. from that laboratory. (Li et al. 2010). The in vitro incubation will be in bpV(pic) (Calbiochem), a PTEN inhibitor, which allows the activation of dormant follicles. and a cell-permeable phospho-peptide (740Y-P) (Tocris) capable of binding to the SH2 domain of the p85 regulatory subunit of PI3K to stimulate enzyme activity. Activated PI3K converts phosphatidylinositol (4, 5)-bisphosphate (PIP2) to phosphatidylinositol (3-5)-trisphosphate (PIP3), whereas the PTEN inhibitor prevents the conversion of PIP3 back to PIP2. Accumulated PIP3, in turn, could stimulate the phosphorylation of Akt and increase the nuclear exclusion of the transcriptional factor Foxo3.