Inflammatory Mechanisms of Dysmenorrhea: Novel Insights from Menstrual Effluent in an Adolescent Cohort

Inflammatory Mechanisms of Dysmenorrhea: Novel Insights from Menstrual Effluent in an Adolescent Cohort | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL BJOG: An International Journal of Obstetrics and Gynaecology This is a preprint and has not been peer reviewed. Data may be preliminary. 11 March 2025 V1 Latest version Share on Inflammatory Mechanisms of Dysmenorrhea: Novel Insights from Menstrual Effluent in an Adolescent Cohort Authors : Chandrashekara N. Kyathanahalli , Frank Tu , and Kevin Hellman 0000-0001-9420-5602 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174167500.02946487/v1 Published BJOG: An International Journal of Obstetrics & Gynaecology Version of record Peer review timeline 452 views 232 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective : To examine how eicosanoid levels in menstrual effluent of adolescents within three years of menarche relate to the severity of menstrual pain. Design : Prospective cohort study. Setting : Community teaching hospital. Population : Adolescents within three years after menarche. Methods : Participants provided a menstrual effluent sample between 4 and 30 months after menarche. Eicosanoids and oxylipins concentrations were measured in the menstrual effluent. We compared effluent concentrations of participants with dysmenorrhea (>0 on a 0-10 scale, n=33) to age-matched pain-free controls (0 on a 0-10 scale, n=18). Main Outcome Measures : Eicosanoid and oxylipins concentrations in menstrual effluent. Results : Participants with dysmenorrhea had higher PGF2α (4.5 [1.6, 8.9] ng/ml, p=0.014) than controls (1.1 [0.07, 4.4] ng/ml). However, differences in PGE2 (7.1 [2.6, 10.1] vs. 3.5 [1.0, 5.1], p=0.053) and 12-HETE (36.3 [23.7, 60.7] vs. 29.6 [13.4, 51.5], p=0.305) were not significant. The correlations between PGF2α (r=0.37, p=0.004) or PGE2 concentration (r=0.28, p=0.046) and menstrual pain intensity were moderate to small. Overall, there were positive correlations between menstrual volume and eicosanoid concentrations (r’s >0.4, p’s< 0.001). Intriguingly, dysmenorrhea participants taking analgesics had a high total content of PGF2α (66.2 [43.0,164.7]) compared to controls (19.1 [6.0,47.5], p=0.04). LC-MS/MS revealed higher concentrations of 12-HETE, 14,15-EET, 15-HETE, 18cdLTB4, LTB4, and PGF2α—and lower 6-kPGF1α—in the effluent of dysmenorrhea participants compared to controls. Conclusions : Elevated PGF2α in adolescents with dysmenorrhea, modest correlations between prostaglandin concentrations and menstrual pain, and the identification of additional oxylipins suggest that inflammatory processes beyond the prostaglandin pathway may contribute to dysmenorrhea. Inflammatory Mechanisms of Dysmenorrhea: Novel Insights from Menstrual Effluent in an Adolescent Cohort Running Title: Eicosanoids in Adolescents with Dysmenorrhea Chandrashekara N. Kyathanahalli 1,2 ; Frank F. Tu 1,2 ; Kevin M. Hellman 1,2 * 1 Department of Obstetrics and Gynecology, Endeavor Health, Evanston, IL, USA 2 Department of Obstetrics and Gynecology, Pritzker School of Medicine, University of Chicago, Chicago, IL, USA Conflict of Interest and Financial Disclosure Statements : F.F.T. receives royalties from Wolters Kluwers and has stock options from Maipl Therapeutics. The remaining authors report no conflict of interest. *Corresponding Author: [email protected] (ORCID ID: 0000-0001-9420-5602) 2650 Ridge Ave, Evanston, IL 60201. (847)-570-3789 Figures: 2 Tables : 4 Supplementary Tables : 2 ABSTRACT: Objective : To examine how eicosanoid levels in menstrual effluent of adolescents within three years of menarche relate to the severity of menstrual pain. Design : Prospective cohort study. Setting : Community teaching hospital. Population : Adolescents within three years after menarche. Methods : Participants provided a menstrual effluent sample between 4 and 30 months after menarche. Eicosanoids and oxylipins concentrations were measured in the menstrual effluent. We compared effluent concentrations of participants with dysmenorrhea (>0 on a 0-10 scale, n=33) to age-matched pain-free controls (0 on a 0-10 scale, n=18). Main Outcome Measures : Eicosanoid and oxylipins concentrations in menstrual effluent. Results : Participants with dysmenorrhea had higher PGF2α (4.5 [1.6, 8.9] ng/ml, p=0.014) than controls (1.1 [0.07, 4.4] ng/ml). However, differences in PGE2 (7.1 [2.6, 10.1] vs. 3.5 [1.0, 5.1], p=0.053) and 12-HETE (36.3 [23.7, 60.7] vs. 29.6 [13.4, 51.5], p=0.305) were not significant. The correlations between PGF2α (r=0.37, p=0.004) or PGE2 concentration (r=0.28, p=0.046) and menstrual pain intensity were moderate to small. Overall, there were positive correlations between menstrual volume and eicosanoid concentrations (r’s >0.4, p’s< 0.001). Intriguingly, dysmenorrhea participants taking analgesics had a high total content of PGF2α (66.2 [43.0,164.7]) compared to controls (19.1 [6.0,47.5], p=0.04). LC-MS/MS revealed higher concentrations of 12-HETE, 14,15-EET, 15-HETE, 18cdLTB4, LTB4, and PGF2α—and lower 6-kPGF1α—in the effluent of dysmenorrhea participants compared to controls. Conclusions : Elevated PGF2α in adolescents with dysmenorrhea, modest correlations between prostaglandin concentrations and menstrual pain, and the identification of additional oxylipins suggest that inflammatory processes beyond the prostaglandin pathway may contribute to dysmenorrhea. Keywords: Menarche, pelvic pain, eicosanoids, menstruation, prostaglandins, oxylipins. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD096332) supported this research. Introduction Dysmenorrhea (menstrual pain) is moderate to severe in more than 40% of those of reproductive age [1,2]. Although some anatomical contributing factors (endometriosis, leiomyomas, and adenomyosis) have been identified as underlying secondary dysmenorrhea, the mechanisms responsible for primary dysmenorrhea are incompletely understood [3] . It has been hypothesized that increased uterine cyclooxygenase-2 activity increases prostaglandin synthesis, which increases uterine contractility, resulting in menstrual pain [3–6]. However, this hypothesis is based on studies with less sensitive methods applied to small sample sizes of older cohorts that likely include secondary dysmenorrhea [7]. Since secondary dysmenorrhea develops later and primary dysmenorrhea emerges within a year post-menarche, early menstrual effluent studies better reveal its underlying mechanisms [8–10]. Although NSAIDs which target COX-2 are the first-line treatment for menstrual pain, many adolescents with dysmenorrhea report inadequate relief, suggesting that additional inflammatory pathways may be involved [11]. Therefore, it is imperative to revisit prostaglandin’s role in menstrual pain and identify other molecules objectively associated with pain severity using contemporary molecular methods in a larger sample size. Together with prostaglandins, arachidonic acid (AA) release caused by endometrial cell breakdown during menses also produces leukotrienes (LTs, from the 5-lipoxygenase pathway) and epoxy eicosatetraenoic acids (EETs, through the cytochrome P450 pathway), which may also influence menstrual pain and inflammatory responses. For example, evidence supporting a role for the 5-lipoxygenase pathway is a study showing that dysmenorrhea effluent has higher levels of LTC4 and LTD4 (which heighten inflammatory and smooth muscle responses) than control participants [12]. A role for the cytochrome P450 pathway is supported by a study showing that elevated 12-hydroxyeicosatetraenoic acid (12-HETE, which triggers inflammation by promoting leukocyte chemotaxis) was higher among participants with menstrual pain [13,14] . Interestingly, 12-HETE is present in concentrations an order of magnitude higher in effluent than prostaglandins, yet it has not been heavily studied to date. Therefore, evaluating uterine eicosanoid synthesis through concentration measurements in menstrual fluid could enhance our understanding of the pathogenesis of dysmenorrhea and contribute to developing new therapeutic options. To determine whether the mechanisms identified in adults—including those with secondary dysmenorrhea—also apply to younger adolescents, we examined eicosanoid profiles in menstrual effluent. We characterized the relevant metabolites in the menstrual effluent, considering that the 5-lipoxygenase and cytochrome P450 pathways could contribute to dysmenorrhea. Additionally, we analyzed the correlation between these factors to quantify how menstrual effluent volume or NSAIDs might impact eicosanoid measurement. Methods 2.1 Study Design The current study’s participants were enrolled in the Early Menstrual Pain Impact on Multisensory Hypersensitivity (EMPATHY) project, which recruited participants before menarche [15,16] . Participants in this substudy had already completed an initial in-person baseline assessment, which involved obtaining parental consent, adolescent assent, a general assessment of experimental pain sensitivity, and comprehensive medical history questionnaires that included demographic information. They submitted a menstrual effluent sample between 4 and 30 months after menarche. The NorthShore University HealthSystem (now Endeavor Health) Institutional Review Board approved this study (EH17-338, September 10, 2018). 2.2 Menstrual Symptom Diary Participants received a written diary to submit along with the menstrual effluent sample. The diary inquired about the timing and intensity of menstrual pain on a 0-10 numerical rating scale (NRS) before inserting the pad/tampon, during the evening while using the pad/tampon, and upon removal. The prospective use of a numerical rating scale is a recognized method in pain research [17] and validated for use in studying dysmenorrhea [18]. In the entire cohort, we verified that there was a strong correlation between diary data and retrospective reports of menstrual pain (r=0.76). Additionally, there was a high correlation between diary data and missed school, social, and physical activities (r=0.74). 2.3 Collection of Menstrual Effluent Participants were given a pre-weighed tampon (Tampax Pearl ®) or pad ( always Pure Cotton ®) and a precisely tared 50 mL tube. They were instructed to use the tampon or pad overnight on the first night following the onset of their menses, to remove it the following morning, and to transfer it to the tared specimen tube. Parents were asked to freeze the samples in their home freezer and return them to the lab within 48 hours. Samples were kept frozen at all times or returned to the lab within 2 hours of removal. On-site, tampons and pads were stored at -80ºC until they were needed for the assay. 2.4 Eicosanoid Extraction from Menstrual Effluent The extraction protocol was modified from Hoefer et al. [13]. Pads or tampons were reweighed and soaked in 150 mL (for pads) or 30 mL (for tampons) of an acid-alcohol mixture (0.1% formic acid in 100% HPLC/spectrophotometric ethanol; #459828 Sigma-Aldrich, Burlington MA). They were placed on an orbital shaker set at 500 rpm for 30 minutes at room temperature and then extracted using a sterile syringe (for tampons) or an autoclaved stainless-steel ricer (for pads). The extracts were centrifuged (Eppendorf, E5810R) at 2,300 Xg for 4 minutes at 4ºC. The supernatant was saved and stored at -80ºC until use. 2.5 Enzyme-Linked Immunosorbent Assays (ELISA) An aliquot (1.5 mL) of the alcoholic extract of menstrual effluent was evaporated to complete dryness in a Speed Vac (SC110, Savant, USA) at room temperature and then reconstituted in 10 mL of Milli-Q water. An aliquot (100 µL) was used to measure the concentrations of Prostaglandin E2 (PGE2, ADI-931-001), Prostaglandin F (PGF2α, ADI-930-069), and 12-HETE (ADI-900-050) by ELISA, following the manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY). The detection limits for these assays were 8.26, 0.98, and 146 pg/mL, respectively. The concentrations of PGE2, PGF2α, and 12-HETE were calculated using a four-point logistic standard curve with the following characteristics: PGE2: r²=0.996; PGF2α: r²=0.983; and 12-HETE: r²=1.00. Each assay exhibited intra- and inter-assay coefficients of variation below 10% and 15%, respectively. 2.6 Hemolysis Interference (HI) Measurements HI was measured by the absorbance of the menstrual effluent using a SpectraMax® M2 spectrophotometer (Molecular Devices, Sunnyvale, CA). A spectral scan of the alcoholic extract and water-reconstituted menstrual effluent was performed in a 96-well plate. Absorbance at 410 nm was recorded at the hemoglobin’s peak absorption wavelength [19]. We validated this technique using blood-extracted menstrual pads in varying cubital blood volumes. The pad’s blood volume strongly correlated with absorbance at 410 nm (r² = 0.91). 2.7 Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) Analysis of Eicosanoids and Oxylipins in Menstrual Effluent We selected samples from five participants who reported the highest levels of overnight menstrual pain (NRS >5) and five participants who reported no overnight menstrual pain for UHPLC-MS analysis. The samples were submitted to Creative Proteomics (Shirley, NY) for analysis. We also submitted spiked samples to assess recovery. Additional analytical details regarding UHPLC-MS are provided in Supplementary Materials Table 1 . 2.8 Statistical Analysis The original study design’s sample size was determined by a power analysis to predict factors related to the development of menstrual pain [15]. For this subset of data, a post hoc sensitivity analysis using G-power 3.1 confirmed that we had 80% power to detect differential levels of PGE2, PGF2α, and 12-HETE when comparing individuals with and without menstrual pain, with an effect size of d > 0.90 and α = 0.05, which is comparable to the existing literature (respective d = 0.94, 1.4, 0.92)[14,20]. However, prior studies were not clear about the criteria used to separate dysmenorrhea participants from controls. For instance, they did not establish cutoffs to distinguish between different levels of menstrual pain severity and participant eicosanoid levels. Therefore, we chose to compare individuals who reported no menstrual pain (rated as 0 on a 0-10 NRS) with those who indicated experiencing menstrual pain (rated as greater than 0 on a 0-10 NRS). All statistical analyses were performed in STATA 13.1 (College Station, TX). The threshold for significance was p<0.05. To estimate total content, raw concentrations of PGE2, PGF2α, and 12-HETE obtained from the ELISA were corrected for differences in extraction volume between pad (150 mL) vs tampon samples (30 mL). Thus, analyses were corrected for volume differences between collection techniques. Before statistical analysis, we also calculated the effluent concentration relative to the weight of menstrual blood collected on a tampon or pad (assuming 1g was equal to 1 mL). Because Shapiro-Wilk Tests confirmed that data were not normally distributed, nonparametric tests were used. Medians and interquartile ranges (IQRs) were calculated for the demographic variables and frequencies (percentages) for categorical variables. Group differences were evaluated using the Kruskal-Wallis test. We calculated Spearman’s correlation coefficient to assess whether average menstrual pain was associated with increased pad/tampon weight or hemolysis (which could influence group differences in eicosanoid content). To verify that concentrations measured with ELISA corresponded with UHPLC-MS measurements, we used Pearson’s correlation coefficient. To analyze and interpret UHPLC-MS data containing a large range of concentrations from multiple biomarkers, data were normalized using their Z-score (mean difference/standard deviation) as recommended [21]. With two-tailed testing, a Z-score of 1.96 or greater would correspond to a significance level of p<0.05. Results 3.1 Demographic and clinical characteristics of the study participants The final sample included 33 participants experiencing dysmenorrhea and 18 pain-free controls ( Figure 1 ). Demographic and menstrual parameters were similar between the groups except for menstrual pain intensity ( Table 1 , p<0.001). Participants with Menstrual Pain have higher PGF 2α Content in Menstrual effluent First, we assessed whether adolescents reporting menstrual pain had greater eicosanoid content than pain-free controls. The median concentrations of PGF2α in menstrual effluent from those with menstrual pain (4.5 [1.6, 8.9]) were significantly higher than in controls (1.1 [0.07, 4.4]; p=0.014, Figure 2 (log2 concentration values presented)). We also calculated the total amount of eicosanoids relative to the used volume of ethanol extract ( Table 2 ). Likewise, participants with menstrual pain had higher levels of total PGF2α than pain-free controls ( Table 2 , p=0.04). Although median concentrations and total levels of PGE2 and 12-HETE were 2 to 3.5 times higher in menstrual pain sufferers than in pain-free controls, the results were not statistically significant ( Table 2 and Figure 2 ). Notably, there was a high coefficient in variation for PGF2α (136%), PGE2 (289%), and 12-HETE (262%) concentrations even after adjusting for group differences. 3.3 Eicosanoid Concentrations were linked to Pain Severity and Effluent Volume/Weight We next examined for meaningful associations between PGE2, PGF2α, and 12-HETE content, the amount of menstrual effluent collected on the tampon or pad, or the extent of hemolysis (due to more blood loss). A higher threshold for significance was used (p<0.01) to account for consideration of multiple comparisons. We found the concentration of eicosanoids was positively associated with the amount of menstrual effluent collected on a tampon or pad (PGE2: r=0.41, PGF2α: r=0.41, and 12-HETE: r=0.58; p’s < 0.01). Except for PGF2α (r=0.31, p=0.027), the presence of hemolytic products significantly influenced detection, and subsequently, the menstrual effluent concentrations of PGE2 (r=0.45, p<0.01) and 12-HETE (r=0.67, p<0.01), and the amount of blood loss and hemolysis (r=0.85, p<0.01). Finally, correlation coefficients were calculated to explore the associations between eicosanoid content in effluent and menstrual pain. Among the three eicosanoids measured, only the correlation between PGF2α and average menstrual pain was significant (r=0.37, p<0.01) ( Supplementary Table 2 ). 3.4 NSAID Usage was not Associated with Reduced Prostaglandin and 12-HETE Content in Dysmenorrhea Participants We then examined whether NSAID use (which is known to inhibit prostaglandin synthesis) was associated with reduced eicosanoid content among participants with dysmenorrhea. Among the 33 dysmenorrhea participants, 11 reported the use of over-the-counter analgesics (such as ibuprofen, naproxen, or acetaminophen). A contrast of characteristics between users and non-users is shown in Table 3 . NSAID non-users utilized tampons or pads had a 4 hour longer median use time compared to users (p<0.05). Thus, results could be biased in favor of finding increased eicosanoid concentrations among those who did not use NSAIDs. However, NSAID users unexpectedly had higher median levels of PGE2, PGF2α, and 12-HETE compared to non-users for pain relief, but only differences for PGF2α were significant. Likewise, NSAID users had 3.3 times higher total median concentrations of PGF2α (p=0.070), 1.3 times higher PGE2 (p=0.445), and 1.1 times higher 12-HETE (p=0.268), but the differences were not significant. 3.5 Identification of Additional Eicosanoids and Oxylipins in Menstrual Effluent To identify other molecules in menstrual effluent that might contribute to pain, we performed UHPLC-MS on five effluent samples from adolescents experiencing the highest intensity of menstrual pain and compared them to samples from those without pain. Of the 76 eicosanoid and eicosanoid-related metabolites assayed, 32 were reliably identified above the assay’s detection limit ( Table 4 ). Among the five participants with dysmenorrhea, we found that six of these 32 eicosanoid and related metabolites were elevated above the threshold for significance (z ≥ 1.96, p ≤ 0.05). Z-scores for significantly elevated metabolites among menstrual pain participants included 12-HETrE (1.97), 14, 15-EET (3.21), 15-HETE (2.8), 18-cdLB4 (2.24), LTB4 (3.0), and PGF2α (2.68). Conversely, 6-kPGF1α was significantly lower in dysmenorrhea participants compared to pain-free controls (2.20, p=0.027). 3.6 Correlation Analysis of Prostaglandins and 12-HETE Concentrations Measured by ELISA and UHPLC-MS/MS. Considering the potential impact of the extraction and analysis method on the study results and interpretation, we evaluated whether LC-MS/MS-based menstrual prostaglandin and 12-HETE measures were comparable to the ELISA-based measures. We calculated Pearson correlations for the samples analyzed for PGE2, PGF2α, and 12-HETE by ELISA and UHPLC-MS/MS methods There was good overall agreement between LC-MS/MS and ELISA methods in the relative concentrations for PGF2α (r =0.93, p < 0.01) and 12-HETE (r = 0.97, p < 0.01). However, the correlation was very weak for the PGE2 (r = 0.27, p = 0.450) concentrations reported by the two analytical methods. 4 Discussion 4.1 Main Findings Within three years of menarche, adolescents reporting any degree of menstrual pain have higher concentrations of PGF2α in their menstrual effluent. The concentration of prostaglandin was correlated with effluent volume, suggesting a role in endometrial sloughing. Paradoxically, dysmenorrhea participants taking NSAIDs (which inhibit prostaglandin synthesis) had higher PGF2α concentrations than those not taking NSAIDs. Further research is needed to determine how to better inhibit PGF2α synthesis, especially at this pivotal point when menstrual pain worsens. Differences in other oxylipin metabolites (12-HETrE, 14, 15-EET, 15-HETE, 18-cdLTB4, LTB4, and 6-kPGF1α) were also linked to menstrual pain. Future research on these molecules could uncover additional targets for treating menstrual pain. 4.2 Interpretation We hypothesized that PGE2 and 12-HETE would be elevated in adolescents with dysmenorrhea. Previous studies indicated that participants with menstrual pain had increased PGE2 in their effluent [14,20,22]. However, the PGE2 ELISA kit we used indicated that it was cross-reactive to PGE1 and PGE3. Additionally, the correlations between the ELISA and UHLPC-MS results suggest that each of our assays exhibited differences in specificity and sensitivity. Likewise, earlier studies using HPLC-UV indicated that 12-HETE (which is found in even higher concentrations than PGF2α) could be elevated among participants with menstrual pain [12,14] . Although our analyses using both ELISA and UHLPC-MS found elevated levels of 12-HETE among participants with menstrual pain, they did not significantly differentiate pain status or correlate with pain severity. We observed a strong correlation between the ELISA and UHLPC-MS assays, supporting the notion that both were specific and sensitive. It is possible that smaller sample sizes and the limited specificity or sensitivity of assays in prior studies contributed to this misclassification. A UHLPC-MS-based targeted assay enabled us to characterize other inflammatory molecules that are differentially expressed among adolescents with menstrual pain. In our study, adolescents with more severe menstrual pain had higher concentrations of several eicosanoids and related metabolites—specifically 12-HETrE, 14,15-EET, 15-HETE, 18-cdLTB4, and LTB4—than their pain-free counterparts. While the elevated presence of LTB4 and 15-HETE in dysmenorrhea aligns with existing literature, the other identified metabolites represent novel findings [12,14] . Mechanistically, these elevated metabolites could contribute to dysmenorrhea: for instance, LTB4 may promote neutrophil recruitment and exacerbate inflammation [23] and 14,15-EET could induce uterine contractions [24] . Moreover, the observed reduction in 6-kPGF1α levels among adolescents with menstrual pain points to a possible disruption in prostacyclin metabolism. Although 6-kPGF1α is not biologically active, increases in its precursor prostacyclin (PGI2) could lead to heightened uterine contractility [25,26] . Overall, these findings suggest that a combination therapy targeting COX-2, LOX, and cytochrome-P450 pathways may provide more effective menstrual pain relief than COX-2 inhibition alone. Potential options to consider in a future clinical trial include Zileuton (a 5-LOX inhibitor) and GSK2256294A (a soluble epoxide hydrolase that would be expected to reduce 14,15-EET) [27] . Although a pilot study investigated the effects of Montelukast (a leukotriene receptor inhibitor) in dysmenorrhea, they failed to demonstrate a superior effect to placebo. However, they never verified that they got a corresponding reduction in effluent inflammatory biomarkers [28] . Additionally, there was only limited use of ibuprofen to reduce COX-2 activity in this trial. Thus, future therapeutic trials should use a combination of LOX and COX inhibitors while simultaneously evaluating the impact of drugs on menstrual effluent biomarkers. 4.3 Clinical Implications Our characterization of prostaglandins and other eicosanoid metabolites in adolescents’ overnight menstrual effluent samples highlights several clinical implications. Previous studies on menstrual flow in early adolescent cohorts relied on pictorial diagrams of flow, which may introduce bias [29,30]. In our study cohort, we previously reported at 4 months, 6.1% self-reported using more than 6 tampons or pads on the heaviest day of their period [16]. The mass of additional effluent in our cohort was typically less than 15g (the maximum a heavy pad or tampon can reliably hold). However, 13.4% of participants had an effluent mass greater than 15g. Thus, the need for multiple heavy tampons or pads during the night in adolescents within three years of menarche may be unusual but not necessarily abnormal. Secondly, patients interested in better managing their menstrual pain should consider using NSAIDs before the start of their period and continuing to use them throughout their period. In our study, participants with dysmenorrhea who used NSAIDs had a 3.3 times higher content of PGF2α than those who did not take NSAIDs. Interestingly, all prior studies on the impact of NSAIDs that reported their ability to reduce PGF2α in menstrual effluent significantly used paradigms involving NSAID use starting either before or at the onset of menses, with sustained usage through day three after menses [7,22,31–33]. One possibility is that it is essential to use NSAIDs early (prophylactically) and also use them continuously, unlike the participants in our cohort. Future research studies should examine whether prophylactic NSAID management provides significant benefits and impacts other candidate mechanisms in menstrual pain. 4.4 Strengths and Limitations Our study is the first to utilize a combination of highly sensitive ELISAs and UHPLC-MS to measure eicosanoid content in one of the most extensive studies of menstrual effluent to date. We also characterized effluent volume in various ways (weight, spectroscopy) to ensure that increased blood flow among participants with dysmenorrhea did not skew the measurement of eicosanoid concentrations. Validating our ability to measure from menstrual pads (instead of tampons) makes effluent studies feasible for other researchers. A key limitation is that the overnight menstrual pain was relatively low. However, our moderate correlation coefficient between PGF2α and menstrual pain indicates that even if we recruited more participants experiencing greater menstrual pain, this would not have significantly affected the findings. Due to cost, we could not apply UHPLC-MS to all biospecimens, making it impossible to reliably correct for multiple comparisons to identify significant differences in the UHPLC-MS analyses. Nevertheless, the UHPLC-MS data correlated well with ELISA samples for PGF2α and 12-HETE; thus, the findings are likely generalizable. It’s possible that the PGE2 data correlated less robustly due to matrix interference related to hemolysis. However, the sample purification in ethanol and formic acid mixture should have mitigated this issue by quickly denaturing enzymes/proteins, removing the matrix, and facilitating eicosanoid extraction [14] . Thus, it would be worthwhile for future studies to confirm differential concentrations of the other eicosanoids in dysmenorrhea participants identified here. 5 Conclusions We confirmed that PGF2α is higher in the menstrual effluent of adolescents with menstrual pain, extending findings in adults to an earlier epoch. Our adult imaging study in adults investigating the effects of naproxen also suggests dysregulation of prostaglandins plays an important role in uterine physiology and pain [34]. However, newly identified oxylipins suggest multiple inflammatory mechanisms beyond prostaglandins. The weak correlation between PGF2α and pain severity (r=0.37) suggests that while prostaglandins contribute to dysmenorrhea, they are unlikely to be the sole mediators of menstrual pain. The identification of elevated leukotrienes and EETs points to a broader inflammatory network influencing pain perception. Intriguingly, NSAID users still exhibited high PGF2α, raising questions about optimal treatment strategies. Future research should explore whether dual inhibition of cyclooxygenase and lipoxygenase pathways could enhance pain relief and provide more effective dysmenorrhea treatments. Authors Roles: CNK: Conception, Experimental Work, Editing, Analysis, Review, Final Approval. FFT & KMH: Obtained funding, Conception, Editing, Data Interpretation, Review, Final Approval. Acknowledgments: We thank Dr. G. F. Gebhart for his assistance with the manuscript. We also thank Ellen Garrison, Sarah Darnell, Kat Dillane, Kaela Harber, Kendra Juliette, and Dina Vavarutsos for technical assistance in running the study visits and collecting the samples. Funding: The Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD096332) supported this research. Conflict of Interest and Financial Disclosure Statements : F.F.T. receives royalties from Wolters Kluwers and has stock options from Maipl Therapeutics. The remaining authors report no conflict of interest. Data Availability: The data underlying this article are available in Open Science Framework, at https://dx.doi.org/10.17605/OSF.IO/ANCJF References [1] Ju H, Jones M, Mishra G. The Prevalence and Risk Factors of Dysmenorrhea. Epidemiologic Reviews 2014;36:104–13. https://doi.org/10.1093/epirev/mxt009.[2] Armour M, Parry K, Manohar N, et al. The Prevalence and Academic Impact of Dysmenorrhea in 21,573 Young Women: A Systematic Review and Meta-Analysis. 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Supplementary Material File (table 1.docx) Download 15.87 KB File (table 2.docx) Download 14.21 KB File (table 3.docx) Download 15.38 KB File (table 4.docx) Download 21.20 KB Information & Authors Information Version history V1 Version 1 11 March 2025 Peer review timeline Published BJOG: An International Journal of Obstetrics & Gynaecology Version of Record 9 Jul 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection BJOG: An International Journal of Obstetrics and Gynaecology Keywords adolescent gynaecology general gynaecology pathology: basic science proteomics reproductive science: prostaglandins translational research Authors Affiliations Chandrashekara N. Kyathanahalli NorthShore University HealthSystem View all articles by this author Frank Tu NorthShore University HealthSystem View all articles by this author Kevin Hellman 0000-0001-9420-5602 [email protected] NorthShore University HealthSystem View all articles by this author Metrics & Citations Metrics Article Usage 452 views 232 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Chandrashekara N. Kyathanahalli, Frank Tu, Kevin Hellman. Inflammatory Mechanisms of Dysmenorrhea: Novel Insights from Menstrual Effluent in an Adolescent Cohort. Authorea . 11 March 2025. DOI: https://doi.org/10.22541/au.174167500.02946487/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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