Analysis of the human endometrial epithelial organoid secretome

A prospective study published in the Proceedings of the National Academy of Sciences (PNAS) has evaluated uterine fluid in the laboratory, thus enabling the analysis of factors influencing uterine fluid composition regulation free from invasive procedures. Such research could have implications for improving pregnancy establishment in women.

Concurrently, the researchers developed a potential model for using precision medicine to improve pregnancy outcomes.

Constantine Simintiras, PhD

“Previous characterizations of human endometrial epithelial organoids (EEOs) from the Universities of Leuven in Belgium and Cambridge in the United Kingdom revealed that EEOs exhibit polarity, whereby apical cell membranes face the center of the organoid, or inward, while the basolateral side faces outward,” said primary author Constantine Simintiras, PhD, a postdoctoral research fellow in interdisciplinary reproduction and health at the University of Missouri in Columbia.

This observation prompted the current investigators to question whether EEOs have the capacity to regulate the composition of intraorganoid fluid (IOF) and extraorganoid fluid (EOF), and if so, “whether the composition of IOF, which is spatially analogous to uterine fluid, resembled that of in vivo uterine fluid,” Simintiras told Contemporary OB/GYN®.

The study, which was conducted at the university’s Thomas Spencer Laboratory from late 2019 to late 2020, used stem cell-derived endometrial organoids to isolate an analogue to uterine fluid in the lab.

The researchers then developed and optimized two complementary strategies for harvesting IOF from three different human donors: aspiration by micromanipulation and high-throughput centrifugation (HTC).

The study demonstrated that IOF is biochemically distinct to EOF. “We are excited by the identification of 17 metabolites in IOF that were absent from EOF,” Simintiras said. “Some of these metabolites are known to be important to pregnancy; the remaining may play currently unknown roles in pregnancy establishment and maintenance.”

A total of 69 metabolites were unique to EOF.

Perhaps the most surprising finding of the study was the detection of 3′-5′-cyclic monophosphate (cAMP) in EOF, according to Simintiras. “The supplementation of cAMP to culture media is required to induce stromal cell decidualization in vitro,” he said. “Although the triggers of decidualization in vivo remain ambiguous, this observation leads us to hypothesize that epithelial basolateral cAMP secretion is a physiological phenotype previously unobservable in vitro and still so in vivo.”

Despite uterine fluid being the site of critical reproductive events, “our understanding of the mechanisms governing uterine fluid formation and composition regulation is poor,” Simintiras said. “This is largely attributable to ethical and technical limitations surrounding human uterine fluid interrogation in vivo, in addition to inadequate cell culture techniques recapitulating uterine fluid formation in vitro.”

Moreover, uterine fluid sampling, by either aspiration or lavage, “remains one of the least invasive methods for collecting material indicative of a woman’s uterine health,” Simintiras said. “As such, our model will hopefully enable us to better understand the mechanisms governing uterine fluid formation and composition regulation, with eventual implications for improving fertility.”

Employing organoids as a model not only averts potential issues with extracting samples during pregnancy, but paves the way for a precision medicine approach to maintaining a healthy pregnancy. By obtaining stem cells from expectant mothers, before they conceive, researchers might be able to study the composition of their uterine fluid to detect concerns, like a deficit in nicotinamide adenine dinucleotide (NAD+), a coenzyme considered crucial for metabolism, and a deficit of which has been linked to birth defects and miscarriage.

Further to advancing the understanding of organoids for improving reproduction, organoids models of different tissues, such as intestinal, continue to be established. “We hope that the technical innovations developed and presented in our study will be applied to wider fields of organoid biology to investigate the formation and regulation of organoid fluid from other secretory cell types,” Simintiras said.

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Disclosure

Simintiras reports no relevant financial disclosures.

Reference

Simintiras CA, Dhakal P, Ranjit C, et al. Capture and metabolomic analysis of the human endometrial epithelial organoid secretome. PNAS. 2021 118 (15) e2026804118. doi:org/10.1073/pnas.2026804118