P-483 Improving in vitro culture of isolated human ovarian primordial-primary follicles by adding adipose derived stem cells
Authors: Camboni A , Cacciottola L , Chiti M.C , Dolmans M.M , Amorim C.A 
Published: June 2022

Abstract

Study question
Could adipose derived stem cells (ADSc) secrete survival- and growth-promoting factors able to improve in vitro culture (IVC) of human isolated follicles?

Summary answer
Co-encapsulation of isolated human primordial-primary follicles with ADSc enhances follicular survival, growth and maturation after 7 days of IVC.

What is known already
The ability to obtain in vitro mature oocyte from earliest follicular stage may help to preserve fertility in female cancer patients.
Unfortunately, human primordial follicles show low rates of growth in vitro. The only study that obtained mature human oocytes from primordial follicle used an IVC multi-step method with a success rate lower than 1% and abnormal polar body.
Recently, several studies have shown that, in mice, ADSc promote survival, antrum formation and oocyte maturation in vitro by secreting factors important for follicular development.
To our knowledge, no IVC studies on human ovarian follicles have been attempted using ADSc.

Study design, size, duration
Fragments of frozen-thawed human ovarian tissue (n = 5) have been mechanically and enzymatically digested to isolate primordial-primary follicles. Eight-to-ten isolated unilaminar follicles have been embedded in alginate-matrigel, half with 20.000 ADSc and half alone. Alginate beads were then cultured in vitro for 7 days. At day (D) 7, we compared isolated follicles co-cultured with ADSc to isolated follicles cultured alone.

Participants/materials, setting, methods
Every two days, IVC medium has been collected to perform hormonal (estradiol, progesteron, anti-Müllerian hormone, androgen, dehydroepiandrosterone) and growth factors measurement (VEGF, bFGF, IGF, HGF) by enzyme-linked immunosorbent assay (ELISA) to study follicle and ADSc interactions. Follicle diameter have been measured at D0 and at D7 of IVC. At D0 and at the end of the IVC (D7), follicle viability (live dead assay) and ultrastructure (transmission electron microscopy [TEM]) have been investigated.

Main results and the role of chance
A total of 373 unilaminar follicles were obtained after enzymatic isolation. At the end of IVC the percentage of viable follicles (with &lt;10% of dead granulosa cells) was found to be significantly higher in follicles co-cultured for 7 days with ADSc (63.3%) compared to follicles in vitro cultured alone (45.5%) (p &lt; 0.01). A significant increase in follicle size was observed in both groups after culture (p &lt; 0.0001). This increase was statistically higher in follicles co-cultured with ADSc than follicles cultured alone (p &lt; 0.005), reflecting better follicle growth in the follicle-ADSc group. Preliminary results by TEM show ADSc did not affect the ultrastructure of isolated follicles after 7 days of IVC.

Limitations, reasons for caution
As demonstrated by our results, ASCs appears to improve IVC conditions and boost follicle activation and survival preserving the follicle ultrastructure. Our study, however, did not investigate the underlying mechanisms behind this improved follicle growth and survival after IVC.

Wider implications of the findings
Co-encapsulation of human unilaminar follicles with ADSCs promotes early-stage ovarian follicle development and survival, possibly because of bidirectional interactions between follicles and ADSCs. This represents a promising first step for fertility preservation in female cancer patients who cannot benefit from cryopreserved ovarian tissue transplantation.

Trial registration number
not applicable