P-805 Gene expression of human endometrial organoids hormonally treated in vitro for 28 days
Authors: Jiang Y , Varshney M , Reyes Palomares A , Inzunza J , Rodriguez-Wallberg K A 
Published: June 2023

Abstract

Study question
Does gene expression of human endometrial organoids (EO) mimic the in vivo response to a 28- days substitutive hormonal treatment?

Summary answer
Gene expression profile of human EO exposed to 28-day hormone treatment showed correspondence with the phases of the menstrual cycle in vivo.

What is known already
The recent development of human EO demonstrated their translational potential for reproductive medicine. Current studies testing functional response to hormones of EO are based on short-term responsiveness to estrogen (E2), progesterone (P4) and differentiation factors up to 10 days. It is unknown how organoids can be used to faithfully reproduce and/or model a complete menstrual cycle of 28 days. High-throughput analyses on endometrium have identified specific markers available for characterizing differentiation and functional aspects related to secretory activity and receptivity to implantation.

Study design, size, duration
Experimental research was carried out including 4 endometrial biopsies donated by women with normal endometrium function, recruited at an academic center for assisted reproduction between 2022-06-15 to 2022-07-28. These endometrial biopsies were processed for organoid derivation and culture. The EO were hormonally treated in vitro and analyzed for gene expression.

Participants/materials, setting, methods
Human endometrial biopsies were enzymatically digested to isolate endometrial glands, embedded in matrigel and cultivated in a spinning bioreactor to achieve organoid formation. Thereafter EO were treated as following: 1) E2: (day 0–28); 2) E2 + P4: E2 (day 0–28), P4 (day 14–28); 3) E2 + P4 + dibutyryl cyclic Adenosine monophosphate (dbcAMP): E2 (day 0–28) + P4 (day 14–28) + dbcAMP (day 14–28) or 4) control group.

Main results and the role of chance
A total of 10 markers were selected for functional analysis and 3 showed a pattern of interest. The target gene expression included Forkhead box O1 (FOXO1), involved in regulating endometrium receptivity in human; progesterone associated endometrium protein (PAEP), a major protein in glandular endometrium secretions; and secreted phosphoprotein 1 (SPP1), an extracellular cytokine that increases in endometrium during the receptive phase.
Gene expression of FOXO1, PAEP and SPP1 showed a significant increase in the organoids exposed to complete hormonal cycle, compared to controls. PAEP and SPP1 gene expression was lower up to day 21 but upregulated to 15-fold relative to non-treated group by day 28. This pattern recapitulates a similar response to the physiological conditions in vivo in the human endometrium. Although gene expression patterns followed similar trends, individual biopsies had slight variations in gene expression along 28-day hormone treatment.
The 28 day-hormone treatment can stimulate EO for recapitulating histological and functional aspects. The dynamics resembled a timely correlation with gene expression changes accounting for some specific markers mostly involved in receptivity, and thus reinforcing the potential application of EO for further research purposes.

Limitations, reasons for caution
In vitro organoid culture conditions may have some influence over responsiveness and gene expression that requires further optimization to reproduce uterine environment in vitro and for other conditions related to infertility in a completely defined culture system.

Wider implications of the findings
Our findings enable the opportunity to expand experimental research over endometrial disorders affecting different stages of the menstrual cycle and also to improve research related to embryo-endometrium interactions.

Trial registration number
This research has been funded by grants from The Swedish Cancer Society, The Swedish Childhood Cancer Foundation, The Cancer Research Funds of Radiumhemmet, The Swedish Cancer Association BRO, the Stockholm County Council (CIMED) and Karolinska Institutet.