Flowing differently: exploratory characterization of menstrual effluent compared with peripheral blood

Our study included samples from 13 healthy participants ( Table 1 ). The mean age of participants was 26 ± 4.4 years old, with the majority of participants being of White race (n=8, 62%). Progesterone and cortisol concentration (ng/mL) were significantly decreased in menstrual effluent compared to peripheral blood ( Figure 3 ). There was no significant difference in concentration (ng/mL) of estradiol and testosterone in peripheral blood compared to menstrual effluent ( Figure 3 ). Among the myeloid cell subsets, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and neutrophils were significantly increased in menstrual effluent compared to peripheral blood ( Figure 4A ). There was no significant difference between menstrual effluent and peripheral blood in content of basophils and classical monocytes ( Figure 4A ). Among the T cell subsets, regulatory T Cells and NKT cells were significantly decreased in menstrual effluent compared to peripheral blood ( Figure 4B ). Memory CD8+ T Cells were significantly increased in menstrual effluent compared to peripheral blood ( Figure 4B ). There was no significant difference between menstrual effluent and peripheral blood in the percentage of CD4+ T Cells, Naive CD4 T Cells, CD4 memory T cells, Total CD8+ T Cells, and Naive CD8+ T cells ( Figure 4B ). Among the NK cell subsets, CD56+CD16- NK Cells, CD56+ CD16+ NK Cells, and CD56++ NK Cells were all significantly increased in menstrual effluent compared to peripheral blood ( Figure 4C ). Among the B cell subsets, naïve B cells, plasmablasts, and memory B Cells were significantly decreased in menstrual effluent compared to peripheral blood ( Figure 4D ). There was no significant difference between menstrual effluent and peripheral blood in content of plasma cells ( Figure 4D ). Concentrations of the following chemokines and cytokines were significantly increased in menstrual effluent compared to peripheral blood: VEGF-A, TARC, MIP-1α, MIP-1β, MIG, MDC, MCP-4, MCP-2, MCP-1, IP-10, IL-7, IL-8, IL-5, IL-33, IL-1α, IL-1RA, IL-18, IL-17A, IL-16, IL-15, IL-12p40, IL-29/IFNlambda, IFN-α2a, and GM-CSF ( Figure 5 ). The concentration of IL-21 was significantly decreased in menstrual effluent compared to peripheral blood ( Figure 5 ). There were no significant differences in concentrations of the following cytokines in menstrual effluent compared to peripheral blood: IFN-γ, IL-10, IL-12p70, IL-13, IL-4, IL-1β, IL-6, TNF-α, TNF-β, IL-2Ra, IL-2, Eotaxin, and Eotaxin-3 ( Figure 5 ). Cytokine findings were further evaluated using a false discovery rate correction, with adjusted q-values reported in Supplemental Table 4 . After correction, GM-CSF, IL-12p40, and IFN-α2a became non-significant.

The study was conducted as shown in Figure 1 . Briefly, women receiving care at the Johns Hopkins Hospital were approached and enrolled if they provided informed consent for blood and menstrual effluent to be collected. Concentration of steroids and cytokines/chemokines were quantified, and immune cell phenotyping was performed. This study was approved by the Johns Hopkins University Institutional Review Board (IRB00302150). Posters advertising the study were placed around the Johns Hopkins campus and surrounding community. Participants contacted the principal investigator if they were interested in participating. Participants were screened for eligibility. Participants were deemed eligible if they met the following criteria: female assigned sex at birth; regular menstrual cycles; non-pregnant, not breastfeeding, no recent delivery within the 3 months preceding collection; no history of miscarriage, abortion, or uterine procedures within the 3 months preceding collection; no recent initiation or discontinuation of hormonal treatment within the 3 months preceding collection; no use of NSAIDs within 3 months, and willingness and ability to travel to the collection site. While many patients are using NSAIDs for menstrual related disorders, we specifically chose to exclude those as prostaglandin synthesis and related pathways are directly impacted by NSAID use and therefore could confound our results. Participants were excluded if they reported a significant history of cardiovascular, endocrine, or menstrual disorders. Informed consent was obtained and signed by both the patient and the principal investigator. Participants were provided a menstrual cup for effluent collection if they did not already own one. If participants had never used a menstrual cup previously, publicly available informational videos were provided by the study team for detailed instruction. Menstrual effluent was collected between the 1 st and 3 rd day of the participant’s menstrual cycle. Participants placed a menstrual cup in the early morning, and menstrual effluent was collected in the afternoon 4–6 hours after placement to ensure adequate volume. At the collection, the participant was led into a private bathroom and given latex gloves and a sterile 50 mL conical tube. The participant was instructed to carefully remove the menstrual cup while wearing gloves – being sure not to spill the contents – and pour the effluent into the conical tube. Media of the following composition was then added to the effluent in a 1 to 1 ratio: RPMI 1640 (500 mL), Glutamax 2mM (5 mL), Penstrep (5mL), FBS 10% (50 mL), Sodium Citrate 0.3% (1.5 g), Pyruvate 1mM (5 mL). The range of menstrual effluent collected from participants was between 1–10 mLs. Peripheral blood was drawn immediately after and collected in 3 acid citrate dextrose tubes. Processing and collection of plasma and mononuclear cells was performed within 12 hours of collection, in tandem and following the same protocol for menstrual effluent and peripheral blood from each participant. Briefly, blood and effluent were centrifuged at 400g for 5 minutes to separate the plasma from the cells. The plasma was isolated and stored at −80°C. Then blood and effluent were mixed with RPMI 1640 (Thermo Fischer Scientific, cat# 11875135) and underlaid with lymphocyte separations media (Corning, cat#25–072). Samples were centrifuged at 400g for 30 minutes. The buffy coat with the mononuclear cells was then collected. The cells from the effluent then underwent an additional processing step where they were strained through a 70µm cell strainer (Corning, cat#431751). Mononuclear cells were washed with RPMI and then cryopreserved in fetal bovine serum (Gibco, cat #A56707–01) supplemented with 10% DMSO (Millipore Sigma, cat# D2653) and stored in liquid nitrogen. Concentrations of testosterone, cortisol, progesterone, and estradiol in peripheral blood and menstrual effluent plasma samples were measured using a competitive multiplex immunoassay kit (MILLIPLEX ® Multi-Species Hormone Magnetic Bead Panel, Millipore-Sigma, cat#MSHMAG-21K) according to manufacturer’s instructions. Briefly, steroids were extracted from samples using an acetonitrile vortex extraction (Sigma-Aldrich, cat#900667) at a 2:3 dilution, samples were centrifuged for 5 minutes at 17,000 × g, supernatants were removed and then dried on a Speed Vac on the lowest heat setting for 5 hours. Samples were reconstituted in assay buffer, and plates were prepared with 25 µL each of assay buffer, HRP-conjugate, and antibody-immobilized beads to each well, then 25 µL of standards, assay quality controls, or sample were added to plates in duplicate. The plate was sealed and protected from light and incubated at 4°C on a rocker for 18 hours. Plates were washed four times using assay wash buffer and a hand-held magnetic plate washer (ThermoFisher, cat#EPX-55555–000). 25 µL of detection antibody was added to each well, and plates were sealed, protected from light, and incubated at room temperature on plate shaker at 350 RPM for one hour. Following incubation, 25 µL of streptavidin-phycoerythrin was added to each well, and plates were sealed, protected from light, and incubated for an additional 30 minutes at room temperature with shaking. Plates were then washed two times using assay wash buffer and the hand-held magnetic plate washer, beads were then resuspended in 100 µL of read buffer (1x Phosphate buffered-saline, 0.05% Tween 20, and 0.1% bovine serum albumin), covered, and placed on a plate shaker for 5 minutes. Plates were then read using a Luminex MagPix ™ immunoassay system, and hormone concentrations were calculated using a 5-parameter logistic curve-fitting method based on the mean fluorescence intensity. Of note, only 10 out of 13 participants produced adequate menstrual effluent volume for steroid quantification. Mononuclear cells were characterized ex vivo by a broad immunophenotyping flow cytometry panel as previously described ( 6 ). Briefly, cells were thawed and stained. Cells were run on a 4 laser (16UV-16V-15B-8R) Cytek Aurora spectral flow cytometer. Manual gating was performed to identify cell populations in effluent and peripheral blood in FlowJo v10 (10.10.0) software. Representative gating is shown in Figure 2A for menstrual effluent and Figure 2B for peripheral blood. Reported as % Live Leukocytes, cells measured are reported in Supplemental Table 1 . We chose to report % Live Leukocytes to accurately reflect the proportion of naïve effector T cells within the viable cell population because naïve T cells are known to populate the endometrium ( 7 ). Menstrual and peripheral blood plasma cytokines and chemokines were measured as previously described ( 8 , 9 ). In brief, concentrations of cytokines and chemokines ( Supplemental Table 2 ) were measured using a custom multiplex kit from Meso Scale Diagnostics (Rockville, MD) according to the manufacturer’s protocol, and data were acquired on a MESO QuickPlex SQ 120. Each plasma sample was measured in duplicate. If an analyte signal was below background, it was set to 0; if detectable, but below the manufacturer’s lower limit of quantification, it was set to the lower limit of detection. If detectable but above the manufacturer’s upper limit of detection, it was set to the upper limit of detection. For cellular frequencies, chemokine and cytokine frequencies, and progesterone hormone concentration, normality of data was assessed using the Shapiro-Wilks test. Data passed normality test at alpha=0.05. If data were found to be normally distributed, significance was tested using paired t-tests. If data were not normally distributed, then Wilcoxon matched pairs signed rank tests were performed to assess statistical significance. Data are presented as mean ± standard error of the mean (SEM). Given the number of comparisons, we applied a Benjamini-Hochberg test for false discovery rate with a threshold of q < 0.01. All statistical analysis was performed in GraphPad Prism 10 and R 4.4.1.

In conclusion, we demonstrate that menstrual effluent is a hormonally and immunologically distinct compartment from peripheral blood, characterized by localized progesterone depletion, reduced cortisol, and unchanged estradiol and testosterone. This endocrine shift is accompanied by a unique immune signature. Future studies should seek to understand the clinical relevance of the immune-endocrine components of menstrual effluent.

This study characterizes immunologic and hormonal differences between menstrual effluent and peripheral blood. Our results are consistent with prior studies showing that the endometrium can produce cytokines during menstruation ( 9 – 14 ). Observed endocrine differences coincided with altered immune cell frequencies and elevated concentrations of cytokines that may be involved in inflammation, tissue remodeling, and repair. The localized decrease of progesterone and cortisol in menstrual effluent is consistent with the post-luteal decline that initiates menstruation. Progesterone normally restricts endometrial inflammation by suppressing NF-κB activation, downregulating COX-2, and limiting transcription of pro-inflammatory cytokines such as IL-8 ( 15 ). Likewise, progesterone withdrawal promotes NF-κB activation, which drives upregulation of IL-6 and promotes local and systemic inflammation. In the endometrium, IL-6 enhances immune cell entry and epithelial activation. Progesterone shifts also increase vascular permeability and facilitate thrombin generation. Downstream, VEGF and other repair-associated pathways have been previously shown to stabilize vasculature and initiate early endometrial tissue regeneration ( 11 ). Disruptions in hormonal regulation, particularly progesterone resistance, have been linked to persistent inflammation in conditions such as endometriosis and heavy menstrual bleeding ( 16 ). Although estradiol levels decline less abruptly during menstruation, estrogen also plays an important role in shaping the uterine immune environment through macrophage recruitment, polarization, and cytokine secretion. Estrogen effects vary heavily depending on receptor distribution and local environmental or cellular cues ( 17 ). While this study was purely correlative in nature and does not imply that inflammatory or immune pathways govern menstruation, this work opens up future avenues related to how the above may be involved in menstrual physiology. The cytokine pattern detected in menstrual effluent in our study aligns with evidence that the endometrium is a secretory tissue with cytokine production that changes across the menstrual cycle. Endometrial epithelial and stromal cells release cytokines and chemokines directly toward the uterine lumen, and this polarized secretion produces local concentrations that differ from those found in peripheral circulation ( 11 , 14 ). These patterns become more pronounced around menstruation as progesterone withdrawal and epithelial breakdown increase the release of several mediators into the uterine cavity ( 2 , 13 ). In our cohort, we observed higher levels of chemokines known to be produced by endometrial cells, including members of the MCP and CXCL families, along with cytokines associated with epithelial stress responses such as IL-33 and IL-15 ( 18 , 19 ). We also observed increased VEGF-A, which is locally produced in endometrial tissue and rises during menses ( 2 , 13 ). For the cytokines that we evaluated, we performed a false discovery rate to decrease risk of type I error due to multiple comparisons and found that certain cytokines that were initially different between menstrual effluent and peripheral blood were no longer significant, including GM-CSF, IL-12p40, and IFN-α2a. This suggest that these cytokines do not show compartmentalization between systemic and uterine immune environments during menstruation ( Supplementary Table 4 ). Immune cell composition in menstrual effluent differed from peripheral blood, further suggesting a menstruation-specific immune environment. Schmitz et al. observed similar CD8+ T cell enrichment in menstrual effluent ( 20 ). Whereas we observed reduced regulatory T cells (Tregs) in menstrual effluent compared to peripheral blood, Shivhare et al. reported Treg accumulation in endometrial tissue during menstruation ( 21 ). This difference may reflect variation between sample types, such as tissue versus effluent, or the capture of different phases of immune activity during menstruation. Although NK cells are known to peak during the secretory phase and decrease following progesterone withdrawal, they remain detectable during menstruation, and several studies have documented their continued presence in menstrual-phase tissue and fluid ( 21 , 22 ). Tindal et al. examined menstrual effluent and peripheral blood in parallel while also including menstrual-phase endometrial biopsies as a secondary study arm. In these endometrial tissue samples, significant reductions in CD56bright NK cells during menstruation were observed ( 3 ). In contrast, our paired effluent and peripheral blood samples demonstrated a relative enrichment of NK cells in menstrual effluent. Prior work has shown that immune cell frequencies differ between menstrual effluent and endometrial tissue, indicating that sampling compartment is a likely contributor to differing NK cell patterns across studies ( 3 , 23 ). Because our study did not measure cytotoxic markers or activating ligands, further work is needed to clarify how NK cell activity changes during menstruation. Our findings extend prior work by Guterstam et al., who documented consistent enrichment of several pro-inflammatory cytokines in menstrual fluid compared with peripheral blood, including IL-1β, IL-6, and TNF-α ( 4 ). By combining an expanded cytokine panel with concurrent hormone profiling, our study provides an integrated view of how local endocrine conditions may correlate with inflammatory pathways during menstruation. This study’s strengths include the use of paired menstrual effluent and peripheral blood samples from the same participant, which circumvents between subject variation. The parallel profiling of cytokines, hormones, and immune cell subsets provides a holistic picture of the menstrual cellular/molecular microenvironment. Several limitations bear mentioning. The cross-sectional design does not allow for temporal resolution of cytokine dynamics across the full menstrual cycle. Furthermore, this was an exploratory study with a sample size sufficient for primary comparisons, but limited for subgroup analyses, and will require subsequent validation. Causal or mechanistic inferences cannot be drawn. Menstrual effluent contains a mixture of blood, tissue fragments, mucus, and cellular debris. Methodologically, we cannot exclude the possibility that sample handling or processing may have influenced the content of the biological specimen. Future studies should seek to collect menstrual effluent as soon as possible after efflux to be able to define hormonal and immunological components with as little manipulation as possible. Lastly, our assays were not all-encompassing, and additional factors known to shape menstrual inflammation such as prostaglandins, matrix metalloproteinases, and local microbiota populations were not assessed ( 3 , 11 ). Future investigations should seek to characterize immune phenotypes of menstrual effluent in disease states such as endometriosis, adenomyosis, polyendocrine metabolic ovary syndrome, and infertility. Menstrual effluent may be useful in the future to predict response to hormonal treatment. Abnormal uterine bleeding is a common cause of etonogestrel implant discontinuation, and we currently lack adequate side effect prediction strategies ( 24 ). Torelli et al. profiled gene expression from endometrial biopsy specimens among etonogestrel contraceptive implant recipients, and correlated this with bleeding profiles ( 25 ). They found that women with lower CXCL1 expression and higher BCL6 and BMP6 expression demonstrated significantly higher risk of unfavorable bleeding at 3 month follow-up. Thus, endometrial or menstrual sampling may provide clinical clues in predicting response to hormonal treatment. Likewise, in progesterone-resistant states such as endometriosis, menstrual effluent analysis may be useful in predicting response to treatment ( 26 , 27 ). Menstrual effluent-based diagnostics could provide a non-invasive tool for clinical assessment of menstrual health, if validated in future prospective studies.

Menstruation is a common physiological process experienced regularly by 1.8 billion reproductive-aged individuals across the world ( 1 ). In those not taking hormonal suppression, irregular menstrual patterns can be a sign of gynecological problems such as infertility/subfertility, anovulatory cycles, polycystic ovarian syndrome, or other endocrinopathies. However, in current clinical practice, refined cellular and molecular analysis of the normal menstrual effluent is lacking. Such knowledge could inform further investigation into how menstrual effluent may change under pathological conditions. Menstruation is characterized by cyclical endometrial breakdown, tissue remodeling, and coordinated local immune activation ( 2 , 3 ). Menstrual effluent, the blood and tissue expelled during menstruation, contains distinct immune and epithelial components from the endometrium, as well as signaling molecules that differ in content and function from those found in peripheral blood ( 2 – 4 ). Sex steroid hormones help orchestrate the cyclical changes of the endometrium by regulating processes as well as influencing immune cell recruitment throughout the menstrual cycle.( 2 ) Cytokines (e.g., chemokines, interleukins, tumor necrosis factors) are small signaling proteins released by immune and stromal cells that mediate inflammation and reflect localized immune activity within the endometrium ( 5 ). Despite growing attention to menstrual effluent as a biospecimen, the hormonal, cellular, and immune profiles remain underexplored. A more direct characterization of menstrual effluent is necessary to understand uterine immune and hormonal signals in their greater biological context. Thus, the aim of this study was to provide an extensive characterization of the immune and hormonal profile of menstrual effluent compared to peripheral blood.