Menstrual Effluent Research Articles

Imaging the dynamics of murine uterine contractions in early pregnancy†.

Uterine muscle contractility is essential for reproductive processes including sperm and embryo transport, and during the uterine cycle to remove menstrual effluent. Even still, uterine contractions have primarily been studied in the context of preterm labor. This is partly due to a lack of methods for studying the uterine muscle contractility in the intact organ. Here, we describe an imaging-based method to evaluate mouse uterine contractility of both the longitudinal and circular muscles in the cycling stages and in early pregnancy. By transforming the image-based data into three-dimensional spatiotemporal contractility maps, we calculate waveform characteristics of muscle contractions, including amplitude, frequency, wavelength, and velocity. We report that the native organ is highly contractile during the progesterone-dominant diestrus stage of the cycle when compared to the estrogen-dominant proestrus and estrus stages. We also observed that during the first phase of uterine embryo movement when clustered embryos move toward the middle of the uterine horn, contractions are dynamic and non-uniform between different segments of the uterine horn. In the second phase of embryo movement, contractions are more uniform and rhythmic throughout the uterine horn. Finally, in Lpar3-/- uteri, which display faster embryo movement, we observe global and regional increases in contractility. Our method provides a means to understand the wave characteristics of uterine smooth muscle in response to modulators and in genetic mutants. Better understanding uterine contractility in the early pregnancy stages is critical for the advancement of artificial reproductive technologies and a possibility of modulating embryo movement during clinical embryo transfers.

Concordance of hemoglobin A1c and reproductive hormone levels in menstrual and venous blood

ObjectiveTo explore whether menstrual blood collected via a modified menstrual pad is a surrogate for venous blood draw in analyzing HbA1c and fertility-associated hormones DesignCross-sectional study SubjectsStudy included 152 female participants who have regular menses, aged 19-50 years old. InterventionsParticipants collected menstrual effluent using a menstrual pad, modified with a removable dried blood spot (DBS) strip. Peripheral blood samples were collected by venipuncture within 60 hours of menstrual pad use. Main Outcome MeasuresMenstrual pad and venous blood draw samples were analyzed for levels of HbA1c, thyroid stimulating hormone (TSH), Follicle Stimulating Hormone (FSH), anti-Mullerian hormone (AMH), and Luteinizing hormone (LH). Correlation between menstrual pad and venipuncture samples was performed by Deming linear regression, and r coefficients were measured using Pearson correlation. ResultsInter-assay variability of menstrual pad DBS sample measurements was <6%. Menstrual HbA1c values were stabilized in the DBS strips through 53 days, and menstrual hormone levels remained stable through 15 days. Menstrual HbA1c levels were highly correlated with venipuncture samples (r=0.96). TSH (r=0.94), AMH (r=0.94), FSH (r=0.91), and LH (r=0.91) also showed high correlation between menstrual strip and venipuncture samples. ConclusionsHbA1c, TSH, AMH, FSH, and LH measurements in menstrual effluent showed high correlation to venous blood samples, supporting use of menstrual effluent as a surrogate sample for hormone testing.

A cross-sectional study comparing the inflammatory profile of menstrual effluent vs. peripheral blood.

Cytokine profiles of peripheral blood and other bodily fluids provide diagnostic indicators for assessing inflammatory processes. Menstrual effluent may provide a noninvasive source of biological material for monitoring cytokine levels in blood and in endometrial tissues. This pilot study investigated the potential of measuring cytokines in menstrual effluent, and compared the cytokine profiles of menstrual versus peripheral blood. Seven healthy donors (aged ≥18 and ≤45 years) collected menstrual effluent on day 2 of menses. Matched peripheral blood samples were collected by venous blood draw on the same day. Levels of 62 cytokines were measured in all samples by 62-plex Luminex assay. Peripheral blood and menstrual effluent cytokine profiles were tenuously correlated (r 2 = 0.26, p < 0.0001), with higher levels detected in menstrual effluent for 48/62 cytokines. Thirty five cytokines were significantly elevated in menstrual effluent compared to peripheral blood samples (IL-8, CCL2, CCL4, LIF, IL-1RA, IL-6, IL-1β, HGF, CCL3, FGF-2, TNF-α, VEGF-A, IL-1α, CXCL1, IL-9, IL-10, EGF, CXCL5, CSF3, EOTAXIN, TGF-α, TRAIL, CXCL10, VEGF-D, IL-12P40, CXCL9, IL-18 RESISTIN, IL-22, IL-21, CSF1, IFN-γ, IL-17A, CXCL12, IL-12p70). Two cytokines (LEPTIN, CSF2) were expressed at significantly lower levels in menstrual effluent compared to peripheral blood. Linear regression of individual cytokines found low predictive power (linear regression p > 0.05) for 53/62 cytokines in menstrual effluent versus peripheral blood. Levels of TGF-β (r 2 = 0.87, p = 0.002) and CCL7 (r 2 = 0.63, p = 0.033) were significantly positively correlated between matched menstrual and peripheral blood samples. In this group of study participants, the cytokine profile of menstrual effluent was quantitatively distinct from peripheral blood, and also characterized by higher levels of inflammatory signaling. This pattern of comparative menstrual blood cytokine profiles points to a need for further studies to evaluate the relationship between peripheral and menstrual blood cytokines in broader populations including both healthy and diseased states.

Single-cell analysis of menstrual endometrial tissues defines phenotypes associated with endometriosis

BackgroundEndometriosis is a common, complex disorder which is underrecognized and subject to prolonged delays in diagnosis. It is accompanied by significant changes in the eutopic endometrial lining.MethodsWe have undertaken the first single-cell RNA-sequencing (scRNA-Seq) comparison of endometrial tissues in freshly collected menstrual effluent (ME) from 33 subjects, including confirmed endometriosis patients (cases) and controls as well as symptomatic subjects (who have chronic symptoms suggestive of endometriosis but have not been diagnosed).ResultsWe identify a unique subcluster of proliferating uterine natural killer (uNK) cells in ME-tissues from controls that is almost absent from endometriosis cases, along with a striking reduction of total uNK cells in the ME of cases (p < 10−16). In addition, an IGFBP1+ decidualized subset of endometrial stromal cells are abundant in the shed endometrium of controls when compared to cases (p < 10−16) confirming findings of compromised decidualization of cultured stromal cells from cases. By contrast, endometrial stromal cells from cases are enriched in cells expressing pro-inflammatory and senescent phenotypes. An enrichment of B cells in the cases (p = 5.8 × 10−6) raises the possibility that some may have chronic endometritis, a disorder which predisposes to endometriosis.ConclusionsWe propose that characterization of endometrial tissues in ME will provide an effective screening tool for identifying endometriosis in patients with chronic symptoms suggestive of this disorder. This constitutes a major advance, since delayed diagnosis for many years is a major clinical problem in the evaluation of these patients. Comprehensive analysis of ME is expected to lead to new diagnostic and therapeutic approaches to endometriosis and other associated reproductive disorders such as female infertility.

T helper 17 axis and endometrial macrophage disruption in menstrual effluent provides potential insights into the pathogenesis of endometriosis

To identify immune cells, cytokines, and immune cell transcriptome in the menstrual effluent (ME) of women with endometriosis compared with that of healthy donors. Live immune cells were isolated from human ME samples and were analyzed by flow cytometry to identify various immune cell populations. Selected cytokines from the same patients were evaluated using multiplex cytokine analyses. The transcriptome of the immune cell population was subsequently profiled using NanoString nCounter's PanCancer Immune panel. Academic institution. Surgically confirmed endometriosis patients (n = 14) and healthy fertile donors (n = 19). None. In-depth immune cell profiling of ME obtained from women with endometriosis compared with that of healthy donors. ME analysis revealed that the number of T helper 17 (TH17) cells was significantly lower in patients with endometriosis compared with that of healthy donors; the number of macrophages was also lower (P=.06) in the former. Multiplex cytokine analysis revealed significantly lower transforming growth factor α in the ME "serum" of patients with endometriosis. Transcriptomic analysis of CD45+ cells revealed 47 differentially expressed genes, mainly associated with the TH17 axis (IL10, IL23A, and IL6), as well as genes associated with macrophage signaling/activation (CD74, CD83, CXCL16, and CCL3). We demonstrate for the first time that the levels of TH17 axis, macrophages, and transforming growth factor α were altered in the ME of women with endometriosis compared with that of healthy donors. These findings shed light on the potential immune pathways that could partly explain the pathogenesis and progression of endometriosis. Future large-scale studies on ME samples are warranted to exploit the use of these markers to study the pathogenesis of endometriosis.

Endometrial microbiota is more diverse in people with endometriosis than symptomatic controls

Endometriosis is a chronic, estrogen-dependent gynecological condition affecting approximately 10% of reproductive age women. The most widely accepted theory of its etiology includes retrograde menstruation. Recent reports suggest the uterus is not sterile. Thus, the refluxed menstrual effluent may carry bacteria, and contribute to inflammation, the establishment and growth of endometriotic lesions. Here, we compared and contrasted uterine bacteria (endometrial microbiota) in people with surgically confirmed presence (N = 12) or absence of endometriosis (N = 9) using next-generation 16S rRNA gene sequencing. We obtained an average of > 9000 sequence reads per endometrial biopsy, and found the endometrial microbiota of people with endometriosis was more diverse (greater Shannon Diversity Index and proportion of ‘Other’ taxa) than symptomatic controls (with pelvic pain, surgically confirmed absence of endometriosis; diagnosed with other benign gynecological conditions). The relative abundance of bacterial taxa enriched in the endometrial microbiota of people with endometriosis belonged to the Actinobacteria phylum (Gram-positive), Oxalobacteraceae (Gram-negative) and Streptococcaceae (Gram-positive) families, and Tepidimonas (Gram-negative) genus, while those enriched in the symptomatic controls belonged to the Burkholderiaceae (Gram-negative) family, and Ralstonia (Gram-negative) genus. Taken together, results suggest the endometrial microbiota is perturbed in people with endometriosis.

Reduced frequency of perforin-positive CD8+ T cells in menstrual effluent of endometriosis patients

Endometriosis is a widespread disease and commonly reduces the life quality of those affected. Scientific literature indicates different underlying immunological changes. Frequently examined tissues are peripheral blood, endometrial tissue and peritoneal fluid. Yet, knowledge on immunological differences in menstrual effluent (ME) is scarce. In this study, between January 2018 and August 2019, 12 women with endometriosis (rASRM classification: stages I-IV) and 11 healthy controls were included. ME was collected using menstrual cups and venous blood samples (PB) were taken. Mononuclear cells were obtained from ME (MMC) and PB (PBMC) and analyzed using flow cytometry. Concentrations of cell adhesion molecules (ICAM-I and VCAM-I) and cytokines (IL-6, IL-8 and TNF-α) were measured using ELISA. CD8 + T cells obtained from ME were significantly less often perforin-positive in women with endometriosis compared to healthy controls. A comparison between MMC and PBMC revealed that MMC contained significantly less T cells and more B cells. The CD4/CD8 ratio was significantly higher in MMC, and Tregs were significantly less frequently in MMC. In ME, T cells and NK cells expressed significantly more CD69. NK cells obtained from ME were predominantly CD56bright/CD16dim and had a lower frequency of perforin + cells compared to PBMC NK cells. Moreover, ICAM-1 plasma levels were significantly reduced in women with endometriosis compared to healthy controls.In conclusion, CD8 + T cells obtained from the ME were significantly less perforin-positive in endometriosis patients indicating a reduced cytotoxic potential. MMC are distinctively different from PBMC and, thus, seem to be of endometrial origin.

Untargeted Metabolomics Reveals that Multiple Reproductive Toxicants are Present in the Endometrium

BACKGROUND AND AIM: Recent epidemiological research focused on the effects of environmental chemicals on the female reproductive system shows mixed results, which may be partially due to differences in exposure assessment, residual confounding from diet or lifestyle factors, or co-exposures to other chemicals. These challenges highlight the need for more precise exposure assessment at the end-organ of interest to accurately understand the adverse reproductive effects of environmental exposures. Our aim was to perform untargeted metabolomic analysis of menstrual blood, a novel biospecimen, to identify environmental toxins present in the endometrium. METHODS: Whole menstrual effluent samples were collected from four women using a menstrual cup. To evaluate the effect of sample processing times, one portion of menstrual effluent was immediately frozen at -20°C for 24hrs and the second portion was kept at 4°C for 24hrs; both samples were then stored at -80°C. Samples were analyzed using untargeted liquid chromatography with high-resolution mass spectrometry (LC-HRMS). To survey the presence of environmental exposures, LC-HRMS chemical profiles were matched to the ToxCast chemical database, which includes 4,557 commonly used commercial chemicals. Select compounds were confirmed using MS/MS and comparison to reference standards. Principal component analysis (PCA) was used to identify differences within and between individuals’ menstrual blood metabolomic profiles, and the influence of the sample processing method. RESULTS:We identified 261 high confidence matches to environmental chemicals, including suspected reproductive toxicants such as phenols, parabens, phthalates, and organochlorides. PCA showed analysis of menstrual blood samples was highly reproducible, with much higher variability between participants than between an individual’s analytical replicates. CONCLUSIONS:Our results show chemical exposures linked to reproductive toxicity and endocrine disruption are present in the endometrium. In addition, our results demonstrate that menstrual blood can be successfully collected and provides a suitable biofluid for untargeted profiling to assess environmental exposures associated with potential adverse reproductive outcomes. KEYWORDS: metabolomics, exposome, biomarkers of exposure, endocrine disrupting chemicals, reproductive outcomes

Comparison of Menstrual Effluent Vascular Endothelial Growth Factor Immunocytochemistry Expression Between Endometriosis and Non-Endometriosis Patients

Objectives: This study aimed to measure the vascular endothelial growth factor (VEGF) expression in menstrual effluent from patients with endometriosis compared to non-endometriosis through immunocytochemical methods. We also measured the degree of pain level, endometrioma cyst size, and infertility status whether it is affected by VEGF expression. Materials and Methods: The present case-control study was conducted in Hasan Sadikin General Hospital, Bandung. Thirty productive-age women diagnosed with endometrioma and 30 productive-age women without endometriosis as the control group were included in this study. Menstrual effluent was taken from the posterior fornix on the second day of menstruation and stained using immunocytochemistry staining for VEGF. Results: The results demonstrated a significant difference between the two study groups in terms of VEGF intensity and histoscore although no difference was found in VEGF distributions between the study groups. The subjects in the endometriosis group had significantly higher VEGF intensity and significantly higher VEGF histoscore compared to the control group. Women with VEGF histoscore more than 6 has 9.33 times risk of developing endometriosis compared to those with lower histoscore. There were no significant correlations between VEGF and pain scale, infertility, and the cyst size. Finally, the cyst size was proportionally related to pain. Conclusions: VEGF distribution and expression in endometriosis women were significantly higher than VEGF levels in non-endometriosis women. Women with menstrual effluent containing higher VEGF levels had more chances of developing endometriosis compared to those with lower VEGF levels. Eventually, larger endometrioma size was proportionally related to higher pain levels in subjects with endometrioma.

Endometrial and Menstrual Blood Mesenchymal Stem/Stromal Cells: Biological Properties and Clinical Application.

A highly proliferative mesenchymal stem/stromal cell (MSC) population was recently discovered in the dynamic, cyclically regenerating human endometrium as clonogenic stromal cells that fulfilled the International Society for Cellular Therapy (ISCT) criteria. Specific surface markers enriching for clonogenic endometrial MSC (eMSC), CD140b and CD146 co-expression, and the single marker SUSD2, showed their perivascular identity in the endometrium, including the layer which sheds during menstruation. Indeed, cells with MSC properties have been identified in menstrual fluid and commonly termed menstrual blood stem/stromal cells (MenSC). MenSC are generally retrieved from menstrual fluid as plastic adherent cells, similar to bone marrow MSC (bmMSC). While eMSC and MenSC share several biological features with bmMSC, they also show some differences in immunophenotype, proliferation and differentiation capacities. Here we review the phenotype and functions of eMSC and MenSC, with a focus on recent studies. Similar to other MSC, eMSC and MenSC exert immunomodulatory and anti-inflammatory impacts on key cells of the innate and adaptive immune system. These include macrophages, T cells and NK cells, both in vitro and in small and large animal models. These properties suggest eMSC and MenSC as additional sources of MSC for cell therapies in regenerative medicine as well as immune-mediated disorders and inflammatory diseases. Their easy acquisition via an office-based biopsy or collected from menstrual effluent makes eMSC and MenSC attractive sources of MSC for clinical applications. In preparation for clinical translation, a serum-free culture protocol was established for eMSC which includes a small molecule TGFβ receptor inhibitor that prevents spontaneous differentiation, apoptosis, senescence, maintains the clonogenic SUSD2+ population and enhances their potency, suggesting potential for cell-therapies and regenerative medicine. However, standardization of MenSC isolation protocols and culture conditions are major issues requiring further research to maximize their potential for clinical application. Future research will also address crucial safety aspects of eMSC and MenSC to ensure these protocols produce cell products free from tumorigenicity and toxicity. Although a wealth of data on the biological properties of eMSC and MenSC has recently been published, it will be important to address their mechanism of action in preclinical models of human disease.

Adenomyosis: Mechanisms and Pathogenesis.

Adenomyosis is a common disorder of the uterus, and is associated with an enlarged uterus, heavy menstrual bleeding (HMB), pelvic pain, and infertility. It is characterized by endometrial epithelial cells and stromal fibroblasts abnormally found in the myometrium where they elicit hyperplasia and hypertrophy of surrounding smooth muscle cells. While both the mechanistic processes and the pathogenesis of adenomyosis are uncertain, several theories have been put forward addressing how this disease develops. These include intrinsic or induced (1) microtrauma of the endometrial-myometrial interface; (2) enhanced invasion of endometrium into myometrium; (3) metaplasia of stem cells in myometrium; (4) infiltration of endometrial cells in retrograde menstrual effluent into the uterine wall from the serosal side; (5) induction of adenomyotic lesions by aberrant local steroid and pituitary hormones; and (6) abnormal uterine development in response to genetic and epigenetic modifications. Dysmenorrhea, HMB, and infertility are likely results of inflammation, neurogenesis, angiogenesis, and contractile abnormalities in the endometrial and myometrial components. Elucidating mechanisms underlying the pathogenesis of adenomyosis raise possibilities to develop targeted therapies to ameliorate symptoms beyond the current agents that are largely ineffective. Herein, we address these possible etiologies and data that support underlying mechanisms.

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

Angiogenic Properties of Menstrual Stem Cells Are Impaired in Women with a History of Preeclampsia

Preeclampsia is a pregnancy-specific disorder defined by the new onset of hypertension and proteinuria after 20 weeks of gestation. Although its precise etiology is still unknown, there is evidence suggesting that it may be a consequence of impaired decidual and stromal cell function. Recently, a stem cell population derived from endometrial tissue and isolated from menstrual effluent called menstrual stem cells (MenSCs) has been identified. MenSCs exhibit important angiogenic and inflammatory properties that may contribute to both normal and pathological complications of implantation and placentation, including preeclampsia. We hypothesized that the angiogenic and inflammatory activity of MenSCs is altered in women who have a past history of preeclampsia and that this phenotype persists postpartum. The primary outcome measures were stromal progenitor cell formation, in vitro induction of endothelial tube formation, and release of proinflammatory cytokines. MenSCs obtained from women with a previous normal or preeclamptic pregnancy displayed similar phenotypic characteristics, tri-differentiation capacity, and proliferation. MenSCs derived from women who had preeclampsia on their previous pregnancy had reduced angiogenic capacity (~30% fewer junctions and nodes, p < 0.05) and stromal progenitor cell formation (<50% measured at a serial dilution of 1 : 10.000, p < 0.05) when compared to controls. In vitro, MenSCs obtained from patients with a history of preeclampsia expressed less endoglin and secreted less VEGF but more IL-6 than controls did. These data are consistent with the hypothesis that the angiogenic and inflammatory properties of MenSCs of women with a previous pregnancy complicated by preeclampsia have reduced angiogenic capacity and are more proinflammatory than those of MenSCs of women with a previous normal pregnancy. This altered phenotype of MenSCs observed following preeclampsia could either be present before the development of the pathology, predisposing the endometrial milieu to and consequently leading to limited vascular remodeling, or be a consequence of preeclampsia itself. The former may afford opportunity for targeted therapeutic intervention; the latter, a putative biomarker for future risk of pregnancy complications.

Analysis of menstrual effluent: diagnostic potential for endometriosis

BackgroundEndometriosis is a chronic and underdiagnosed disease which affects 5–10% of women of childbearing age and is characterized by growth of endometrial tissue outside of the uterus, most often in the peritoneal cavity. Delay in diagnosis is a major problem for management of this disorder, and treatment is often not initiated until the disease has progressed for many years. Although the exact etiology of endometriosis remains unknown, retrograde menstruation is recognized as a common underlying factor leading to the deposit of menstrual effluent (ME) into the peritoneal cavity. Differences in the cellular biology and genetics of the cells within ME are therefore likely to explain why endometriosis develops in only a subset of women.MethodsPatients with and without endometriosis were consented to provide ME. ME was analyzed by flow cytometry for CD45- and CD45+ cell populations or used to isolate stromal fibroblast cells. ME-derived stromal fibroblast cells were assessed using decidualization assays following the addition of cAMP and IGFBP-1 concentrations in the culture supernatants were measured by ELISA. In addition, RNA was collected and analyzed by RNA-Seq and qPCR for markers of decidualization and to identify differentially expressed genes in ME-derived stromal fibroblast cells obtained from controls and subjects with endometriosis (±cAMP).ResultsFlow cytometry analysis of cell subsets within the CD45+ fraction of ME revealed a significant decrease in the number of uterine NK cells in endometriosis patients compared with controls (p < 0.01). No other significant differences within either the CD45+ or CD45- cell populations were observed. Most strikingly, ME-derived stromal fibroblast cells cultured from endometriosis subjects showed impaired decidualization potential compared with controls. Highly significant differences in decidualization response were detected by measuring IGFBP-1 production at multiple time points after cAMP stimulation (p = 0.0025 at 6 h; p = 0.0045 at 24 h; p = 0.0125 at 48 h). RNA-Seq and qPCR analyses were used to identify genes differentially expressed by ME-derived stromal fibroblast cells obtained from endometriosis and control subjects.ConclusionsMenstrual effluent can be useful for investigating the pathobiology of endometriosis and for developing a non-invasive diagnostic for endometriosis which may lead to earlier and more effective treatments for this common disorder.

Perioperative Evaluation in Herlyn-Werner-Wunderlich Syndrome

Herlyn-Werner-Wunderlich syndrome is defined by uterine didelphys, obstructed hemivagina, and ipsilateral renal agenesis. We report the presentation, radiographic findings, and outcomes of three patients with Herlyn-Werner-Wunderlich syndrome who underwent surgical management at a tertiary care academic medical center. Two patients were diagnosed by magnetic resonance imaging and one by ultrasonography. All three underwent successful transvaginal septoplasty and drainage of the hematocolpos and hematometra. One required septoplasty revision with temporary vaginal stent placement. Herlyn-Werner-Wunderlich syndrome is a rare condition. The most urgent concern is the obstruction of menstrual effluent. Unlike an isolated transverse vaginal septum, the vaginal septum with Herlyn-Werner-Wunderlich syndrome is parallel to the patent vaginal canal, less pliable, and closer to the cervix. These anatomical differences result in pronounced distortion with significant diagnostic and surgical challenges.

Identification of Cells with Colony-Forming Activity, Self-Renewal Capacity, and Multipotency in Ovarian Endometriosis

Endometriosis, the growth of endometrial tissue outside the uterine cavity, is a common gynecological disorder affecting 10% to 15% of women in their reproductive years. Retrograde menstrual shedding containing endometrial stem/progenitor cells has been postulated to be involved in its pathogenesis. In this study, we identified putative endometriotic stem/progenitor cells by their colony-forming potential, self-renewal capacity, and multipotency. Purified epithelial and stromal cells isolated from ovarian endometriotic cysts formed large and small colony-forming units (CFUs) in clonogenic assay. The colony-forming activity of epithelial and stromal cells was found to differ greatly between autologous endometrium and ovarian endometrioma samples. The large CFUs could propagate more than the small CFUs. The endometriotic epithelial small CFUs expressed epithelial markers (epithelial cell adhesion molecule, cytokeratin, and α6 integrin); only occasional large CFUs expressed α6 integrin. Aside from the expression of fibroblast markers, stromal CFUs also expressed three somatic stem cell markers: sal-like 4, CD133, and Musashi-1. Endometriotic stromal cells derived from large CFUs could differentiate into four mesenchymal lineages when cultured in the respective inducing-media, as determined by histochemical staining and RT-PCR of lineage specific markers. These findings demonstrate that ovarian endometrioma contains a subset of cells displaying somatic stem cell properties.

Bacterial endotoxin in menstrual effluent as a case of spontaneous abortion and preterm labor

Bacterial endotoxin in menstrual effluent as a case of spontaneous abortion and preterm labor

Reduced levels of VEGF-A and MMP-2 and MMP-9 activity and increased TNF-α in menstrual endometrium and effluent in women with menorrhagia

Heavy regular menstrual periods (menorrhagia) are an important cause of ill health in women and remain the leading indication for hysterectomy. Abnormalities of the endometrial blood vessels are among the possible causes of this condition. Many different factors affect endothelial cell growth, function and vessel remodelling. We sought to determine whether the levels of vascular endothelial growth factor-A (VEGF-A), tumour necrosis factor-alpha (TNF-alpha), matrix metalloproteinase (MMP)-2 and MMP-9 and soluble VEGF receptor-1 (VEGF-R1) were altered in the menstrual effluent of women with objective menorrhagia. We have also quantitated the VEGF-A mRNA in the menstruated endometrium. We recruited 37 women and determined their menstrual blood loss (MBL) over two cycles and collected menstrual effluent during the 2nd day of bleeding for 4 h. There was no difference in the total level of VEGF-A, and neither latent MMP. However, the concentration of VEGF-A was significantly reduced in the women with menorrhagia, as was the VEGF-A mRNA level. In addition, the active forms of both MMPs were markedly reduced and the total sVEGF-R1 as well as the TNF-alpha content were increased. This is the first study to show abnormalities of factors important for endothelial cell behaviour in the endometrium of women with menorrhagia. This may underlie the disordered vessel structure and/or function in this condition.

Menstrual effluent in endometriosis shows no difference in volume, VEGF-A, MMP2 and MMP9 or sFLT

Since retrograde menstruation is considered a key event in the aetiology of endometriosis, this study sought to determine whether the menstrual effluent of women with this condition is different from that of those with a normal pelvis. As the amount of blood lost during menstruation is thought to be higher in this group, measured objective menstrual blood loss (MBL) was measured. In addition, factors enhancing both ectopic implantation of endometrium and its subsequent growth (by establishing a neo-vasculature) were chosen for study. Our hypothesis was that they are increased in the menstrual effluent of women with endometriosis. The study showed that at the time of menstruation, there is no difference in MBL or in the volume of menstrual effluent between women with endometriosis and those with a normal pelvis at laparoscopy. In addition, vascular endothelial growth factor-A (VEGF-A) message and protein, soluble truncated receptor sVEGF-R1 (sFLT), matrix metalloproteinase (MMP) 2 and MMP9 activities were also shown to be similar between the two groups. It is concluded that the enhanced expression of VEGF-A and MMP in the peritoneal fluid and ectopic lesions of endometriotic patients may be a secondary event, resulting from an innate difference in peritoneal and systemic factors rather than in the endometrium, causing an abnormal peritoneal response to menstrual debris and facilitating its ectopic implantation.

Molecular characterization of soluble factors from human menstrual effluent that induce epithelial to mesenchymal transitions in mesothelial cells

We have studied menstrual effluent in order to identify soluble menstrual factors that induce epithelial to mesenchymal transitions (EMT) in mesothelial cells. A variety of molecules, such as nitric oxide and its reaction products, proteases (i.e. matrix metalloproteinases, plasmin) and proteins and/or peptides (i.e. growth factors: b-fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, transforming growth factor-beta; cytokines: interleukin 1 beta, tumour necrosis factor-alpha [TNF-alpha]) may be involved in this process. We have demonstrated that TNF-alpha is involved in EMT, whereas the other molecules are not. Biochemical analysis has shown that the inducing menstrual factors are heat-labile molecules, are uncharged at neutral pH, have a molecular weight between 50-70 kDa (or are bound in complexes of that size) and are eluted in the albumin fraction during gel filtration chromatography. Further analysis of this fraction by using proteomics and mass spectrometry has led to the identification of alpha-enolase and haemoglobin whose inhibition partially prevents EMT. When antibodies against TNF-alpha, alpha-enolase and haemoglobin are combined, EMT is almost completely inhibited. Thus, the candidates for soluble menstrual factors that induce mesothelial EMT are TNF-alpha, alpha-enolase and haemoglobin.