Derivation of mature oocytes from human primordial follicles The majority of ovarian follicles stay dormant at the primordial stage for decades in women and the depletion of this dormant pool leads to menopause. Due to genetic and environmental influences, some women suffer from premature ovarian failure/insufficiency associated with a diminishing follicle reserve. Under physiological conditions, uncharacterized intraovarian mechanisms activate ~1,000 dormant primordial follicles to initiate growth per month, whereas the remaining follicles stay quiescent for years or decades. Once recruited into the growing pool, primordial follicles continue growth to the early antral stage with minimal loss. At the early antral stage, select follicles respond to gonadotropins and one preovulatory follicle release the mature oocyte for fertilization. Studies using mutant mice indicated that oocyte-specific deletion of the PTEN (Tumor-suppressor phosphatase with TENsin homology) gene or the downstream transcriptional factor Foxo3 promoted the growth of all primordial follicles. The PTEN gene encodes a phosphatase enzyme that negatively regulates the phosphoinositol 3-kinase (PI3K) and Akt signaling pathway. Deletion of PTEN in the oocyte increases the phosphorylation of Akt and the nuclear exclusion of the downstream Foxo3 proteins, leading to the activation of all dormant primordial follicles. Taking advantage of the availability of PTEN inhibitors, we activated primordial follicles in mice. Neonatal mouse ovaries exposed transiently to PTEN inhibitors in vitro showed marked increases in follicle growth after transplantation into the kidney capsule of FSH-treated adult recipients. Mature oocytes could be retrieved from these ovaries for IVF and blastocyst formation. Our preliminary data also demonstrated the efficacy of the PTEN inhibitor in activating dormant HUMAN primordial follicles in cortical strips donated by a cancer patient to yield mature oocytes. The Fertility and Cancer program at Stanford have stored frozen human ovarian cortical strips containing mainly primordial follicles from cancer patients. Here, we propose to incubate human follicles in vitro with PTEN inhibitors and paracrine factors, followed by transplantation into immune-deficient mice to optimize the follicle activation approach for the derivation of mature human oocytes. Chromosomal integrity will be investigated for individual mature oocytes generated after transplantation to determine the safety of the present procedure. In addition, we will use a two-step in vitro culture approach to treat mouse neonatal ovaries and human cortical strips with PTEN inhibitor and ovarian paracrine growth factors for the activation of dormant follicles, followed by the in vitro promotion of their growth to secondary and preantral follicles using stage-specific paracrine factors. During the second step, we will isolate preantral follicles and culture them with diverse ovarian paracrine factors to promote their development into antral follicles containing mature oocytes. We will further investigate the chromosomal integrity of individual mature oocytes derived from in vitro cultures and further amplify RNA from individual mature oocytes for DNA microarray analyses to reveal transcriptome changes. Although fertility is compromised in patients with premature ovarian failure and peri-menopausal women, their ovaries still contain many primordial follicles. The present in vitro follicle activation approach, followed by auto- transplantation, could allow retrieval of functional mature oocytes for infertile women with diminishing ovarian reserve and for cancer patients. Future development of a two-step culture approach to allow the development of primordial follicles to the antral stage, followed by the generation of mature oocytes, could have important clinical implications for the treatment of ovarian infertility in diverse population of women.
Patients with premature ovarian failure or female cancer patients following chemo- or radiation therapy suffer from the loss of ovarian germ cells and infertility. The present application deals with the use of enzyme inhibitors and key ovarian growth factors to initiate the growth of dormant human ovarian follicles for subsequent derivation of mature oocytes. Once optimized, the present approach could allow the activation of residual primordial follicles from patients with premature ovarian failure or peri-menopausal women to generate mature oocytes for infertility treatment. Frozen ovaries from cancer patients before chemo- or radiation therapy could also be used to generate mature oocytes for infertility treatment.
