Abstract
Research Question: What are the true benefits, if any, of disrupting the Hippo signaling pathway and stimulating the Akt pathway in xenotransplanted human ovarian tissue using an in vitro activation (IVA) approach?Design: Human ovarian tissue was retrieved from 18 young patients by laparoscopy and grafted to 54 severe combined immunodeficient mice. The experiment was conducted using fresh ovarian tissue (group I; n = 6 women), slow-frozen-thawed ovarian tissue (group II; n = 6 women), and vitrified-warmed ovarian tissue (group III; n = 6 women). Slow-freezing and vitrification procedures were performed according to Gosden's and Kawamura's protocols, respectively. The tissue (fresh, slow-frozen, and vitrified) was fragmented into small cubes (1 × 1 × 1 mm) to disrupt the Hippo signaling pathway and cultured or not in IVA medium for 48 h with Akt stimulators (PI3K stimulator and PTEN inhibitor), before being transplanted to the mice. All the grafts were maintained for 28 days.Results: (1) Follicular density: Follicular density decreased in all groups after transplantation, most significantly in the vitrification group. Culture with IVA had no impact. (2) Follicle activation: Addition of PI3K stimulator and PTEN inhibitor for 48 h prior to grafting did not significantly change the proportion of primordial follicles in any of the groups (fresh, slow-frozen, or vitrified tissue) compared to 48 h of control culture without these molecules. Particularly, vitrification and culture in IVA medium yielded no benefits in terms of growing follicle percentages or follicle proliferation rates. The large proportion of growing follicles in the vitrified tissue group after grafting may have been responsible for the higher rate of atresia.Conclusion: We were unable to demonstrate any significant benefits of cutting ovarian tissue into small cubes and applying IVA with Akt stimulators. The association of vitrification and transplantation was actually found to be the most deleterious combination with respect to the follicle reserve, and even worse when culture with Akt stimulators was performed.
Highlights
Human folliculogenesis is a complex process regulated by endocrine hormones and paracrine and autocrine factors of oocyte and granulosa cell (GC) origins [1]
While most primordial follicles remain in a state of arrest due to dormancy factors, selected primordial follicles initiate growth, developing to the primary stage under the control of serine/threonine kinases through two signaling pathways: protein kinase B (Akt) and mammalian target of rapamycin [1]
Disruption of this pathway, combined with stimulation of Akt signaling by treatment with phosphatase and tensin homolog (PTEN) inhibitors and/or PI3K stimulators, was shown to promote growth of preantral follicles [2, 4,5,6]
Summary
Human folliculogenesis is a complex process regulated by endocrine hormones and paracrine and autocrine factors of oocyte and granulosa cell (GC) origins [1]. Development of preantral follicles is curbed by the inhibitory Hippo signaling pathway [1, 3], so disruption of Hippo signaling promotes secretion of downstream growth factors capable of stimulating follicle growth [1, 2] Disruption of this pathway (which occurs with ovarian tissue fragmentation), combined with stimulation of Akt signaling by treatment with phosphatase and tensin homolog (PTEN) inhibitors and/or PI3K stimulators, was shown to promote growth of preantral follicles [2, 4,5,6]. In 2016, Zhai et al reported a live birth in a series of 14 patients with pre-mature ovarian insufficiency (POI) after removal of one ovary, 2 days of IVA, and reimplantation beneath the serosa of one of the fallopian tubes [8]
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