Functional assessment of human endometrial organoid responsiveness to a sequentially controlled hormonal treatment mimicking menstrual cycle

To assess the response of human endometrium organoids (hEOs) subjected to a controlled hormonal treatment mimicking the female menstrual cycle. Four endometrial biopsies donated by women with normal endometrium function were enzymatically digested to isolate endometrial glands and stromal cells, embedded in matrigel and cultivated in a spinning bioreactor to achieve organoid formation. Thereafter hEOs were sequentially treated as following: 1) Estradiol (E2) day 0–28; 2) E2 day 0–28 + Progesterone (P4) day 14–28; 3) E2 day 0–28 + P4 day 14–28 + dibutyryl cyclic Adenosine monophosphate (dbcAMP) day 14–28; 4) control group. Confocal imaging through immunofluorescence (IF) and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to detect and quantify pre-selected markers of interest for evaluating human endometrial development. After normalization, ΔΔCt fold changes between treatment group and control group were compared using unpaired t-test, and p<0.05 were considered significant (two-sided). Analyses were conducted with Graphpad Prism 9 software. Expression of progesterone receptor increased with E2 and E2+P4 treatments compared to control group, response was consistent even after long term (28 days) hormone treatment. The hEOs In group 2 and 3 exhibited glandular structures and positiveness of progesterone associated endometrium protein (PAEP) in epithelial cells indicating differentiation towards secretory phase. Secretory phase marker PAEP and secreted phosphoprotein 1 (SPP1) as well as the ciliated cells marker Alpha-tubulin were expressed in group 2 and group 3. SPP1 gradually increased from group 2 reaching maximum in group 3, suggesting that response to progesterone and cAMP are both crucial in inducing secretory endometrium features in hEOs models, closely resembling functional response of human endometrium during this phase. The hEOs respond to hormone treatment in vitro mimicking physiological changes occurring in the human endometrium in a 28 days’ menstrual cycle. Treated hEOs could recapitulate histological and functional features of endometrium in vivo.