Menstrual fluid endometrial stem/progenitor cell and supernatant protein content: cyclical variation and indicative range

Abstract

STUDY QUESTION Does natural variation exist in the endometrial stem/progenitor cell and protein composition of menstrual fluid across menstrual cycles in women? SUMMARY ANSWER Limited variation exists in the percentage of some endometrial stem/progenitor cell types and abundance of selected proteins in menstrual fluid within and between a cohort of women. WHAT IS KNOWN ALREADY Menstrual fluid is a readily available biofluid that can represent the endometrial environment, containing endometrial stem/progenitor cells and protein factors. It is unknown whether there is natural variation in the cellular and protein content across menstrual cycles of individual women, which has significant implications for the use of menstrual fluid in research and clinical applications. STUDY DESIGN, SIZE, DURATION Menstrual fluid was collected from 11 non-pregnant females with regular menstrual cycles. Participants had not used hormonal medications in the previous 3 months. Participants collected menstrual fluid samples from up to five cycles using a silicone menstrual cup worn on Day 2 of menstrual bleeding. PARTICIPANTS/MATERIALS, SETTING, METHODS Menstrual fluid samples were centrifuged to separate soluble proteins and cells. Cells were depleted of red blood cells and CD45+ leucocytes. Menstrual fluid-derived endometrial stem/progenitor cells were characterized using multicolour flow cytometry including markers for endometrial stem/progenitor cells N-cadherin (NCAD) and stage-specific embryonic antigen-1 (SSEA-1) (for endometrial epithelial progenitor cells; eEPC), and sushi domain containing-2 (SUSD2) (for endometrial mesenchymal stem cells; eMSC). The clonogenicity of menstrual fluid-derived endometrial cells was assessed using colony forming unit assays. Menstrual fluid supernatant was analyzed using a custom magnetic Luminex assay. MAIN RESULTS AND THE ROLE OF CHANCE Endometrial stem/progenitor cells are shed in menstrual fluid and demonstrate clonogenic properties. The intraparticipant agreement for SUSD2+ menstrual fluid-derived eMSC (MF-eMSC), SSEA-1+ and NCAD+SSEA-1+ MF-eEPC, and stromal clonogenicity were moderate-good (intraclass correlation; ICC: 0.75, 0.56, 0.54 and 0.52, respectively), indicating limited variability across menstrual cycles. Endometrial inflammatory and repair proteins were detectable in menstrual fluid supernatant, with five of eight (63%) factors demonstrating moderate intraparticipant agreement (secretory leukocyte protein inhibitor (SLPI), lipocalin-2 (NGAL), lactoferrin, follistatin-like 1 (FSTL1), human epididymis protein-4 (HE4); ICC ranges: 0.57–0.69). Interparticipant variation was limited for healthy participants, with the exception of key outliers of which some had self-reported menstrual pathologies. LARGE SCALE DATA N/A. There are no OMICS or other data sets relevant to this study. LIMITATIONS, REASONS FOR CAUTION The main limitations to this research relate to the difficulty of obtaining menstrual fluid samples across multiple menstrual cycles in a consistent manner. Several participants could only donate across <3 cycles and the duration of wearing the menstrual cup varied between 4 and 6 h within and between women. Due to the limited sample size used in this study, wider studies involving multiple consecutive menstrual cycles and a larger cohort of women will be required to fully determine the normal range of endometrial stem/progenitor cell and supernatant protein content of menstrual fluid. Possibility for selection bias and true representation of the population of women should also be considered. WIDER IMPLICATIONS OF THE FINDINGS Menstrual fluid is a reliable source of endometrial stem/progenitor cells and related endometrial proteins with diagnostic potential. The present study indicates that a single menstrual sample may be sufficient in characterizing a variety of cellular and protein parameters across women’s menstrual cycles. The results also demonstrate the potential of menstrual fluid for identifying endometrial and menstrual abnormalities in both research and clinical settings as a non-invasive method for assessing endometrial health. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Australian National Health and Medical Research Council to C.E.G. (Senior Research Fellowship 1024298 and Investigator Fellowship 1173882) and to J.E. (project grant 1047756), the Monash IVF Research Foundation to C.E.G. and the Victorian Government’s Operational Infrastructure Support Program. K.A.W., M.L.D.-T., S.G.S. and J.E. declare no conflicts of interest. C.E.G. reports grants from NHMRC, during the conduct of the study; grants from EndoFound USA, grants from Ferring Research Innovation, grants from United States Department of Defence, grants from Clue-Utopia Research Foundation, outside the submitted work. CEF reports grants from EndoFound USA, grants from Clue-Utopia Research Foundation, outside the submitted work.

Comparison of Organoids from Menstrual Fluid and Hormone-Treated Endometrium: Novel Tools for Gynecological Research

“…The exclusion criterion for participation was the use of hormonal contraceptives and/or other exogenous hormonal treatment within the last 3 months. Menstrual fluid samples were collected using a silicon menstrual cup (Lunette, Juupajoki, Finland), which was worn by the participants for 4-6 h on day 2 of menstruation, as previously described [9]. Samples were transferred into 50 mL polypropylene tubes by the participants, transported at 4 • C, and received by laboratory research staff within 2 h of collection.…”

“…The human MF samples were digested as previously described [9] with the exception that the collagenase 2 digestion was halted when clusters of 5-10 epithelial cells could be seen microscopically (usually after 10 min). Clusters were then enriched for by epithelial cell adhesion molecule (EpCAM) magnetic beading (4 beads/cell, CELLection Epithelial Enrichment kit, 162.03, Invitrogen) before seeding at 7000 clusters per well in Matrigel ® , as described previously [5][6][7].…”

“…Excess clusters were cryopreserved (30% fetal bovine serum (FBS), 10% Dimethyl sulfoxide (DMSO), Advanced DMEM:F12, 10 uM Rock Inhibitor) for the subsequent generation of MFO upon thawing. MF supernatant, CD45 + leukocytes, Sushi domain-containing protein 2 + (SUSD2 + ) eMSC, and SUSD2 − stromal cells were isolated as described [1,9] and were cryopreserved (90% FBS, 10% DMSO) for subsequent experiments.…”

“…The expression of cell surface markers EpCAM, Neural-cadherin (N-cadherin, NCAD, a marker of endometrial epithelial progenitor cells, eEPC), and Stage Specific Embryonic Antigen-1 (SSEA-1, a putative eEPC marker) were determined by flow cytometry as previously described [9]. Briefly, Passage 3 day 7 organoids were dissociated to clusters mechanically [7] and subsequently to single cells with TryplE Express Enzyme (12604-021, Invitrogen).…”

“…Menstrual fluid represents a novel biofluid with the potential for diagnosis and disease modeling [9]. We have shown that a range of menstrual fluid cells and proteins have minimal variation from cycle to cycle [9] and that endometrial stem and progenitor cells can be found in menstrual fluid and the pelvic cavity and therefore may play a role in endometriosis [9,10]. Therefore, we proposed that menstrual fluid could be a non-invasive source of endometrial organoids that may enable the population-wide study of early disease.…”