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.

Endometrial organoids (EMO) are an important tool for gynecological research but have been limited by generation from (1) invasively acquired tissues and thus advanced disease states and (2) from women who are not taking hormones, thus excluding 50% of the female reproductive-aged population. We sought to overcome these limitations by generating organoids from (1) menstrual fluid (MF; MFO) using a method that enables the concurrent isolation of menstrual fluid supernatant, stromal cells, and leukocytes and (2) from biopsies and hysterectomy samples from women taking hormonal medication (EMO-H). MF was collected in a menstrual cup for 4–6 h on day 2 of menstruation. Biopsies and hysterectomies were obtained during laparoscopic surgery. Organoids were generated from all sample types, with MFO and EMO-H showing similar cell proliferation rates, proportion and localization of the endometrial basalis epithelial marker, Stage Specific Embryonic Antigen-1 (SSEA-1), and gene expression profiles. Organoids from different disease states showed the moderate clustering of epithelial secretory and androgen receptor signaling genes. Thus, MFO and EMO-H are novel organoids that share similar features to EMO but with the advantage of (1) MFO being obtained non-invasively and (2) EMO-H being obtained from 50% of the women who are not currently being studied through standard methods. Thus, MFO and EMO-H are likely to prove to be invaluable tools for gynecological research, enabling the population-wide assessment of endometrial health and personalized medicine.