Creation of a Bioengineered Ovary: Isolation of Female Germline Stem Cells for the Repopulation of a Decellularized Ovarian Bioscaffold.

Ovarian failure is the most common cause of infertility and affects about 1% of young women. One innovative strategy to restore ovarian function may be represented by the development of a bioprosthetic ovary, obtained through the combination of tissue engineering and regenerative medicine.We here describe the two main steps required for bioengineering the ovary and for its ex vivo functional reassembling. The first step aims at producing a 3D bioscaffold, which mimics the natural ovarian milieu in vitro. This is obtained with a whole organ decellularization technique that allows the maintenance of microarchitecture and biological signals of the original tissue. The second step involves the use of magnetic activated cell sorting (MACS) to isolate purified female germline stem cells (FGSCs). These cells are able to differentiate in ovarian adult mature cells, when subjected to specific stimuli, and can be used them to repopulate ovarian decellularized bioscaffolds. The combination of the two techniques represents a powerful tool for in vitro recreation of a bioengineered ovary that may constitute a promising solution for hormone and fertility function restoring. In addition, the procedures here described allow for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Hedgehog pathway inhibition causes primary follicle atresia and decreases female germline stem cell proliferation capacity or stemness.

BACKGROUND: Follicle depletion is one of the causes of premature ovarian failure (POF) and primary ovarian insufficiency (POI). Hence, maintenance of a certain number of female germline stem cells (FGSCs) is optimal to produce oocytes and replenish the primordial follicle pool. The mechanism that regulates proliferation or stemness of FGSCs could contribute to restoring ovarian function, but it remains uncharacterized in postnatal mammalian ovaries. This study aims to investigate the mechanism by which inhibiting the activity of the hedgehog (Hh) signaling pathway regulates follicle development and FGSC proliferation. METHODS AND RESULTS: To understand the role of the Hh pathway in ovarian aging, we measured Hh signaling activity at different reproductive ages and the correlation between them in physiological and pathological mice. Furthermore, we evaluated the follicle number and development and the changes in FGSC proliferation or stemness after blocking the Hh pathway in vitro and in vivo. In addition, we aimed to explain one of the mechanisms for the FGSC phenotype changes induced by treatment with the Hh pathway-specific inhibitor GANT61 via oxidative stress and apoptosis. The results show that the activity of Hh signaling is decreased in the ovaries in physiological aging and POF models, which is consistent with the trend of expression levels of the germline stem cell markers Mvh and Oct4. In vitro, blocking the Hh pathway causes follicular developmental disorders and depletes ovarian germ cells and FGSCs after treating ovaries with GANT61. The proliferation or stemness of cultured primary FGSCs is reduced when Hh activity is blocked. Our results show that the antioxidative enzyme level and the ratio of Bcl-2/Bax decrease, the expression level of caspase 3 increases, the mitochondrial membrane potential is abnormal, and ROS accumulate in this system. CONCLUSIONS: We observed that the inhibition of the Hh signaling pathway with GANT61 could reduce primordial follicle number and decrease FGSC reproductive capacity or stemness through oxidative damage and apoptosis.

Oogonial Precursor Cell-Derived Autologous Mitochondria Injection to Improve Outcomes in Women With Multiple IVF Failures Due to Low Oocyte Quality: A Clinical Translation.

BACKGROUND: Mitochondrial dysfunction has been suggested as a major cause of age-induced decline in oocyte quality. In the past, donor oocyte cytoplasmic transfer showed some success but was abandoned due to the concerns with heteroplasmy. Recent studies indicated presence of oogonial precursor cells (OPCs) in the human ovary, which could be an autologous source of "healthy mitochondria." We sought to investigate the clinical efficacy of OPC-derived autologous mitochondrial injection (AMI) to improve oocyte quality in women with multiple in vitro fertilization (IVF) failures. METHODS: The OPCs were isolated from laparoscopically obtained ovarian cortical pieces by cell sorting using a monoclonal anti-vasa homolog (anti-DDX) antibody. They were then disrupted and mitochondria were isolated. Reconstituted mitochondria were injected into each oocyte during intracytoplasmic sperm injection. Paired comparisons were made between the first failed cycles and the post-AMI cycles. RESULTS: Of the 15 women undergoing ovarian stimulation, 2 were canceled and 3 decided to pool oocytes for later AMI. In remaining 10 (mean age 34.7 ± 4.1), AMI significantly improved fertilization rates (49.7 ± 31.3 vs 78.3 ± 18.9; P = .03) with a trend for better embryo grades (2.3 ± 0.3 vs 3.1 ± 0.7; P = .08). Four of 10 women conceived after single frozen embryo transfer and 3 after confirmation of diploidy via array comparative genomic hybridization (aCGH) (clinical pregnancy/embryo transfer = 4/10). CONCLUSION: These data show encouraging results for AMI in comparison to previous failed IVF cycles.