Development and characterization of human ovarian spheroids

Details

Supervisors

Andrade Amorim, Christiani ; Tavares Sousa, Maria João

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Premature ovarian insufficiency (POI) is characterized by the premature exhaus;on of ovarian func;on before the age of 40, leading to hormonal imbalances and health complica;ons such as osteoporosis, cardiovascular disorders, and infer;lity. Conven;onal hormone replacement therapies (HRTs), based on the con;nuous administra;on of estradiol and/or progesterone, effec;vely mi;gate menopausal symptoms, but pose long-term risks, including increased risks of breast cancer and stroke. Our study aimed to develop a cell-based HRT alterna;ve by crea;ng a spheroid model of human theca (TCs) and granulosa cells (GCs) to mimic the human ovarian follicle steroidogenic compartment and its interac;on with the hypothalamic- pituitary-ovarian (HPO) axis. GCs were collected from 40 pa;ents undergoing in vitro fer;liza;on, while TCs were differen;ated from postmenopausal ovarian stromal cells of 7 organ donors. These TCs and GCs were assembled into mul;cellular spheroids, with spheroids containing only GCs (G-only spheroids) serving as controls, and cultured for 15 days to analyze morphology and hormone secre;on to iden;fy the op;mal ;me for embedding into a hydrogel. Live/DeadTM assay was employed to assess cellular viability before embedding and at the end of the culture, and morphology and steroid produc;on were quan;fied. Upon collec;on, GCs expressed markers such as CYP19A1, and FSHR, while differen;ated TCs expressed CYP17A1 and CD13. Interes;ngly, G-only spheroids showed increased area and reduced roundness over ;me, contras;ng with the consistent morphology of mul;cellular spheroids. Hormonal quan;fica;on confirmed a robust func;onal ac;vity of spheroids at this stage. Spheroids were embedded in PEGylated fibrin to assess long-term viability. No significant decrease in viability was observed prior to embedding and aner 30 days of culture. All spheroid types showed an increase in 17ß-estradiol levels, followed by an accentuated decrease at day 12 of culture. Progesterone levels remained stable throughout the culture period. While PEGylated fibrin supported ini;al cell viability, its limita;ons became evident through significant cell invasion and a decline in estradiol secre;on over ;me. Alginate was used as an alterna;ve hydrogel to address these challenges. This hydrogel successfully preserved the spherical morphology of the embedded spheroids. These engineered ovarian follicle spheroids may offer a promising alterna;ve for HRT, providing a physiologically relevant in vitro model for studying follicular development and advancing innova;ve approaches in HRT research.