Targeting osteopontin alleviates endometriosis and inflammation by inhibiting the RhoA/ROS axis and achieves non-invasive in vitro detection via menstrual blood.

STUDY QUESTION: How does osteopontin (OPN) in endometriosis ectopic stromal cells (EESCs) participate in the pathogenesis of endometriosis and achieve non-invasive detection in vitro? SUMMARY ANSWER: Targeted OPN regulates endometriosis's necroptosis and inflammatory state by inhibiting the RhoA/reactive oxygen species (ROS) axis, thereby alleviating endometriosis and enabling non-invasive detection of menstrual blood in vitro. WHAT IS KNOWN ALREADY: Endometriosis is a chronic inflammatory disease. Recent studies have shown that OPN plays an important role in disease progression by regulating cell death and inflammation. STUDY DESIGN, SIZE, DURATION: The study included 20 patients diagnosed with endometriosis (confirmed by laparoscopy and histology) and 10 controls without endometriosis. Endometriotic stromal cells were isolated from endometrial samples, while menstrual blood endometrial cells (MESCs) were isolated from menstrual blood. These cells were then cultured in vitro and utilized in subsequent experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: OPN expression in EESCs was assessed using inflammatory factor sequencing, immunohistochemical staining (IHC), quantitative real-time PCR (qRT-PCR) analysis, and Western blotting (WB). The biological behavior of OPN and its effects on inflammatory factors were examined using EdU, wound-healing, Transwell, and ELISA assays. Necroptosis in EESCs and its impact on inflammatory factors were detected through qRT-PCR, WB, and Calcein-AM/PI fluorescence assays. The examination of mitochondrial stress in EESCs involved the use of the Mitochondrial Membrane Potential (ΔΨm) Assay, ROS detection, and Calcein-AM Loading/cobalt chloride Quenching. qRT-PCR, WB, and other experiments were conducted to verify the regulation of necroptosis and inflammatory factor levels in EESCs by OPN through the RhoA/ROS axis. Knockdown of OPN and its inhibitory effect on endometriosis lesion size were confirmed using AAV9 virus, IHC, qRT-PCR, WB, and other experiments. Additionally, OPN expression in MESCs was detected using transcriptome sequencing, RT-PCR, WB, and other experiments. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro assays demonstrated a significant upregulation of OPN in EESCs, and the knockdown of OPN effectively inhibited necroptosis and the release of inflammatory factors. OPN inhibited necroptosis and inflammatory factor release by mediating RhoA-dependent ROS production and blocking mixed lineage kinase domain-like protein phosphorylation at the cell membrane. In vivo, targeting of OPN can inhibit the growth of endometriosis lesions. Clinically, OPN was also significantly upregulated in the menstrual blood of patients with endometriosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to limitations in obtaining surgical specimens, our study primarily involved collecting endometriosis tissues from women during the proliferative and secretory phases of the menstrual cycle. We observed a significant overexpression of OPN in the samples used for our investigation. However, the expression of OPN in endometriosis tissues during the intermenstrual phase remains unknown. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the pivotal role of the OPN/RhoA/ROS axis in the regulation of necroptosis and the release of inflammatory factors. OPN knockdown exerts a therapeutic effect in vivo, and the high expression detection of OPN in menstrual blood in vitro. In summary, targeting OPN provides possibilities for the treatment and detection of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071626), the Zhejiang Province Public Welfare Technology Application Research Project (LGF21H040010), and the Clinical Research project of the Second Affiliated Hospital of Wenzhou Medical University (1010293). The authors have no conflicts of interest.

HGF Secreted by Menstrual Blood-Derived Endometrial Stem Cells Ameliorates Non-Alcoholic Fatty Liver Disease Through Downregulation of Hepatic Rnf186.

Mesenchymal stem cells (MSCs) have been demonstrated to protect against fatty liver diseases, but the mechanism is still not clear. Menstrual blood-derived endometrial stem cells (MenSCs) are a substantial population of MSCs that can be obtained in a noninvasive manner. In the present study, we investigated the therapeutic effects and underlying mechanisms of MenSC transplantation in mouse models of diet-induced nonalcoholic fatty liver disease (NAFLD). The results revealed that MenSCs markedly promoted hepatic glycogen storage and attenuated lipid accumulation after transplantation. We further identified Rnf186 as a novel regulator involved in MenSC-based therapy for NAFLD mice. Rnf186 deficiency substantially inhibited high-fat diet-induced insulin resistance and abnormal hepatic glucose and lipid metabolism in mice. Mechanistically, Rnf186 regulated glucose and lipid metabolism through the AMPK-mTOR pathway. More importantly, hepatocyte growth factor (HGF) is identified as the key functional cytokine secreted by MenSCs and decreases the expression of hepatic Rnf186. HGF deficient MenSCs cannot attenuate glucose and lipid accumulation after transplantation in NAFLD mice. Collectively, our results provide preliminary evidence for the protective roles of HGF secreted by MenSCs in fatty liver diseases through downregulation of hepatic Rnf186 and suggest that MenSCs or Rnf186 may be an alternative therapeutic approach/target for the treatment of NAFLD.

Quantitative evaluation of endometrium-expressed mRNAs for the purpose of discriminating between menstruation and traumatic vaginal injury in sexual assault cases.

In sexual assault cases, it is crucial to discriminate between peripheral blood and menstrual blood to provide evidence for vaginal intercourse with traumatic injury. In this study, the menstrual blood mRNA markers progestagen-associated endometrial protein (PAEP), matrix metallopeptidase 7 (MMP7), and left-right determination factor 2 (LEFTY2) were evaluated by quantitative RT-PCR (RT-qPCR) for the discrimination of menstrual blood from peripheral blood and vaginal fluid. As a result, all markers with cutoff delta cycle quantification (ΔCq) values were specifically determined in menstrual blood among forensically relevant body fluids. Even though the changes in the expression levels of each marker differed during the menstrual cycle, all markers were determined to be positive in most of the randomly collected menstrual blood samples that were analyzed. Additionally, the markers with proposed cutoff ΔCq values could discriminate between menstrual blood and peripheral blood-mixed vaginal fluid samples. The determination of positive markers was less affected by storage temperature under dry conditions than under wet conditions, while PAEP was detectable in samples stored below room temperature under wet conditions. The detectability of PAEP was considered to be the result of its higher expression level compared with MMP7 and LEFTY2. In conclusion, menstrual blood markers for the RT-qPCR procedure evaluated in this study were highly specific for menstrual blood. The proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract. In particular, PAEP is expected to be applicable to forensic casework samples because of its high specificity and robustness.

(Un)Reliable detection of menstrual blood in forensic casework - evaluation of the Seratec® PMB test with mock samples.

The identification of the type of body fluid in crime scene evidence may be crucial, so that the efforts are high to reduce the complexity of these analyses and to minimize time and costs. Reliable immunochromatographic rapid tests for specific and sensitive identification of blood, saliva, urine and sperm secretions are already routinely used in forensic genetics. The recently introduced Seratec® PMB test is said to detect not only hemoglobin, but also differentiate menstrual blood from other secretions containing blood (cells) by detecting D-dimers. In our experimental set-up, menstrual blood could be reliably detected in mock forensic samples. Here, the result was independent of sample age and extraction buffer volume. It was also successfully demonstrated that all secretions without blood cells were negative for both, hemoglobin (P) and D-dimer (M). However, several blood cell-containing secretions/tissues comprising blood (injury), nasal blood, postmortem blood and wound crust also demonstrated positive results for D-dimer (M) and were therefore false positives. For blood (injury) and nasal blood, this result was reproduced for different extraction buffer volumes. The results of this study clearly demonstrate that the Seratec® PMB test is neither useful nor suitable for use in forensic genetics because of the great risk of false positive results which can lead to false conclusions, especially in sexual offense or violent acts.

Enhanced therapeutic potential of Flotillins-modified MenSCs by improve the survival, proliferation and migration.

Menstrual blood-derived endometrial stem cells (MenSCs) have attracted increasing interest due to their excellent safety, and lack of ethical dilemma as well as their ability to be periodically obtained in a noninvasive manner. However, although preclinical research as shown the therapeutic potential of MenSCs in several diseases, their poor cell survival and low engraftment at disease sites reduce their clinical efficacy. Flotillins (including Flot1 and Flot2) are implicated in various cellular processes, such as vesicular trafficking, signal transduction, cell proliferation, migration and apoptosis. In this study, we aimed to determine the effects of Flotillins on MenSCs survival, proliferation and migration. Our experimental results show that MenSCs were modified to overexpress Flot1 and/or Flot2 without altering their intrinsic characteristics. Flot1 and Flot2 co-overexpression promoted MenSC viability and proliferation capacity. Moreover, Flot1 or Flot2 overexpression significantly promoted the migration and inhibited the apoptosis of MenSCs compared with the negative control group, and these effects were stronger in the Flot1 and Flot2 gene co-overexpression group. However, these effects were significantly reversed after Flot1 and/or Flot2 knockdown. In conclusion, our results indicate that Flot1 and Flot2 overexpression in MenSCs improved their proliferation and migration and inhibited their apoptosis, and this might be an effective approach to improve the efficiency of cell-based therapies.

Extracellular vesicle-transmitted miR-671-5p alleviates lung inflammation and injury by regulating the AAK1/NF-κB axis.

Mesenchymal stem cells regulate remote intercellular signaling communication via their secreted extracellular vesicles. Here, we report that menstrual blood-derived stem cells alleviate acute lung inflammation and injury via their extracellular vesicle-transmitted miR-671-5p. Disruption of this abundantly expressed miR-671-5p dramatically reduced the ameliorative effect of extracellular vesicles released by menstrual blood-derived stem cells on lipopolysaccharide (LPS)-induced pulmonary inflammatory injury. Mechanistically, miR-671-5p directly targets the kinase AAK1 for post-transcriptional degradation. AAK1 is found to positively regulate the activation of nuclear factor κB (NF-κB) signaling by controlling the stability of the inhibitory protein IκBα. This study identifies a potential molecular basis of how extracellular vesicles derived from mesenchymal stem cells improve pulmonary inflammatory injury and highlights the functional importance of the miR-671-5p/AAK1 axis in the progression of pulmonary inflammatory diseases. More importantly, this study provides a promising cell-based approach for the treatment of pulmonary inflammatory disorders through an extracellular vesicle-dependent pathway.

Raman Spectroscopy for the Time since Deposition Estimation of a Menstrual Bloodstain.

Forensic chemistry plays a crucial role in aiding law enforcement investigations by applying analytical techniques for the analysis of evidence. While bloodstains are frequently encountered at crime scenes, distinguishing between peripheral and menstrual bloodstains presents a challenge. This is due to their similar appearance post-drying. Raman spectroscopy has emerged as a promising technique capable of discriminating between the two types of bloodstains, offering invaluable probative information. Moreover, estimating the time since deposition (TSD) of bloodstains aids in crime scene reconstruction and prioritizing what evidence to collect. Despite extensive research focusing on TSD estimations, primarily in peripheral bloodstains, a crucial gap exists in determining the TSD of menstrual bloodstains. This study demonstrates how Raman spectroscopy effectively analyzes biological samples like menstrual blood, showing similar aging patterns to those of peripheral blood and provides proof-of-concept models for determining the TSD of menstrual blood. While this work shows promising results for creating a universal model for bloodstain age determination, further testing with more donors needs to be conducted before the implementation of this method into forensic practice.

The Concept behind the Suitability of Menstrual Blood-Derived Stem Cells for the Management of Vaginal Atrophy among BRCA Mutation Carriers after RRSO.

Risk-reducing bilateral salpingo-oophorectomy (RRSO) is recommended for breast cancer gene 1 (BRCA1) and 2 (BRCA2) mutation carriers. A major consequence of RRSO is surgical menopause associated with severe menopausal symptoms, mostly genitourinary complaints. Due to the inherent breast cancer risk, estrogen-based therapies are generally avoided in these patients. So far, the non-hormonal approaches available are not efficient to successfully treat the disabling vaginal atrophy-related symptoms. In regenerative medicine, mesenchymal stem cells (MSC) are the most frequently used cell type due to their remarkable and regenerative characteristics. Therapies based on MSC have revealed positive outcomes regarding symptoms and signs associated with vaginal atrophy by promoting angiogenesis, vaginal restoration, and the proliferation of vaginal mucosa cells. Menstrual blood-derived stem cells (MenSC) are a novel source of MSC, with promising therapeutic potential directly linked to their high proliferative rates; low immunogenicity; non-invasive, easy, and periodic acquisition; and almost no associated ethical issues. In this review, we update the current knowledge and research regarding the potential value of previously preserved MenSC in the therapy of vaginal atrophy among BRCA mutation carriers subjected to RRSO.

Menstrual Blood Stem Cells-Derived Exosomes as Promising Therapeutic Tools in Premature Ovarian Insufficiency Induced by Gonadotoxic Systemic Anticancer Treatment.

Gonadotoxicity resulting from systemic and locoregional cancer treatments significantly threatens women's reproductive health, often culminating in premature ovarian insufficiency. These therapies, particularly alkylating agents and ionizing radiation, induce DNA damage and apoptosis in ovarian follicles, leading to infertility, amenorrhea, and estrogen deficiency, which exacerbate risks of osteoporosis and cardiovascular diseases. Existing fertility preservation methods do not prevent immediate ovarian damage, underscoring the need for innovative protective strategies. Menstrual blood-derived stem cells (MenSC) and their extracellular vesicles (EV) present promising regenerative potential due to their therapeutic cargo delivery and pathway modulation capabilities. Preclinical studies demonstrate that MenSC-derived EV ameliorate premature ovarian insufficiency by inhibiting granulosa cell apoptosis, promoting angiogenesis, and activating pivotal pathways such as SMAD3/AKT/MDM2/P53. However, comprehensive research is imperative to ensure the safety, efficacy, and long-term effects of MenSC-derived EV in clinical practice. In this review, we update the current knowledge and research regarding the use of MenSC-derived EV as a novel therapeutic weapon for ovarian regeneration in the context of gonadotoxicity induced by systemic anticancer treatment.

Menstrual blood-derived stromal cells: insights into their secretome in acute hypoxia conditions.

BACKGROUND: Despite constant advances in regenerative medicine, the closure of chronic wounds is still challenging. Therapeutic approaches using locally administered MSCs have been considered a promising option. However, the viability of these cells is seriously threatened by acute hypoxic stress linked to wound healing. In this work, we aimed to study the tolerance of Menstrual blood-derived stromal cells (MenSCs) to acute hypoxia and their therapeutic paracrine effect. METHODS: Isolated MenSCs were phenotypically characterized and evaluated in terms of proliferation, viability, and gene expression, under acute hypoxia (AH) compared with conventional cultured condition or normoxia (N). A step further, the secretome of MenSCs under acute hypoxia was analyzed with respect to their miRNAs content and by in vitro functional assays. For the analysis of differences between the two groups, Student's t-test was performed and one-way ANOVA and Tukey's multiple comparisons test for multiple groups were used. RESULTS: Our results revealed that the viability of MenSCs was not affected under acute hypoxia, although proliferation rate slowed down. Gene analysis revealed 5 up-regulated (BNIP3, ANGPTL4, IL6, IL1B, and PDK1) and 4 down-regulated genes (IDO1, HMOX1, ANGPTL2, and HGF) in AH compared to N. Global gene expression analysis revealed a decrease in the gene ontology functions of migration and wound response with respect to the normoxic condition. In contrast, functions such as angiogenesis were enriched under the AH condition. Regarding the secretome analysis, two miRNAs involved in angiogenic processes (hsa-miR-148a-3p and hsa-miR-378a-3p), were significantly up-expressed when compared to the normoxic condition, being MYC gene, the unique target of both. Functional assays on HUVECs revealed a potential pro-angiogenic capacity of MenSCs cultured in both oxygen conditions (N and AH) based on the wound closure and tube formation results of their released paracrine factors. However, when compared to normoxia, the paracrine factors of MenSCs under acute hypoxia slightly reduced the proliferation, migration, and in vitro wound closure of HUVECs. CONCLUSIONS: MenSC exhibited a good survival capacity under acute hypoxic conditions as well as beneficial properties applicable in the field of tissue regeneration through their secretome, which makes them a potential cell source for wound healing interventions.

Biopsy-free profiling of the uterine immune system in patients with recurrent pregnancy loss and unexplained infertility.

RESEARCH QUESTION: What are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) and patients with unexplained infertility (uINF)? DESIGN: Prospective study including 46 healthy controls, 28 RPL and 11 uINF patients. A feasibility study compared lymphocyte compositions of endometrial biopsies and menstrual blood collected during the first 48 h of menstruation in seven controls. In all patients, peripheral and menstrual blood from the first and subsequent 24 h were analysed separately by flow cytometry, focusing on the main lymphocyte populations and natural killer (NK) cell subsets. RESULTS: The first 24 h of menstrual blood resembles the uterine immune milieu as tested by endometrial biopsy. RPL patients showed significantly higher menstrual blood CD56+ NK cell numbers than controls (mean ± SD: 31.13 ± 7.52% versus 36.73 ± 5.4%, P = 0.002). Menstrual blood CD56dimCD16bright NK cells within the CD56+ NK cell population were decreased in RPL (16.34 ± 14.65%, P = 0.011) and uINF (15.7 ± 5.91%, P = 0.02) patients versus control (20.42 ± 11.53%). uINF patients had the lowest menstrual blood CD3+ T cell counts (38.81 ± 5.04%, control versus uINF: P = 0.01) and cytotoxicity receptors NKp46 and NKG2D on CD56brightCD16dim cells were higher in uINF (68.12 ± 11.84%, P = 0.006; 45.99 ± 13.83%, P = 0.01, respectively) and RPL (NKp46: 66.21 ± 15.36%, P = 0.009) patients versus controls. RPL and uINF patients had higher peripheral CD56+ NK cell counts versus controls (11.42 ± 4.05%, P = 0.021; 12.86 ± 4.29%, P = 0.009 versus 8.4 ± 3.5%). CONCLUSIONS: Compared with controls, RPL and uINF patients had a different menstrual blood-NK-subtype profile, indicating an altered cytotoxicity. In future studies, this non-invasive analysis might enable identification and monitoring of patients receiving immunomodulatory medications.

Comparison of menstruation recovery after ultrasound-guided suction curettage in patients with cesarean scar pregnancy pretreated using high-intensity focused ultrasound and uterine artery embolization.

OBJECTIVES: To investigate the menstruation recovery after two therapeutic regimens, high-intensity focused ultrasound (HIFU) or uterine artery embolization (UAE) combined with ultrasound-guided suction curettage, for premenopausal women with cesarean scar pregnancy (CSP). MATERIALS AND METHODS: Seventy patients with CSP treated with HIFU followed by ultrasound-guided suction curettage and 63 patients with CSP treated with UAE followed by ultrasound-guided suction curettage in our hospital were retrospectively reviewed. The treatment parameters and the menstrual blood loss (MBL) volume at 6 months after the two therapeutic regimens were compared between the two groups. RESULTS: There was no significant difference in treatment results between the two groups. With regard to postoperative MBL volume, 5 out of 70 patients in the HIFU group and 22 out of 63 patients in UAE group exhibited a reduction in MBL, respectively. Multivariate logistic regression revealed that hypomenorrhea was more likely to occur in the UAE group than in the HIFU group (UAE group vs. HIFU group; OR 11.328, 95% CI 3.158-55.871; p < 0.001). CONCLUSION: Compared to UAE, HIFU has less influence on postoperative MBL volume, which may be a fertility-sparing option for patients with CSP.

The effect of vitamin D on the severity of dysmenorrhea and menstrual blood loss: a randomized clinical trial.

BACKGROUND: Primary dysmenorrhea is considered as one of the women's main problems during reproductive age. The present study aimed to investigate the effect of vitamin D on the severity of dysmenorrhea and menstrual blood loss. METHODS: This double-blind, randomized, placebo-controlled trial, was performed on 84 single female college students between 18 and 25 years old who living in dormitories. Students with primary dysmenorrhea and vitamin D deficiency were divided into experimental (n = 42) and control (n = 42) groups. Five days before the putative beginning of their next menstrual cycle, the experimental group received 300,000 IU vitamin D (50,000 IU, two tablets every 8 h), and the control group received a placebo (oral paraffin). The effects of the supplement on the severity of dysmenorrhea and menstrual blood loss were evaluated one cycle before and during two successive cycles. Using the visual analog scale (VAS), verbal multidimensional scoring system (VMS), and pictorial blood assessment chart (PBLAC) questionnaires. Fisher's exact, Chi-square, independent sample t-test and repeated measurements were used. RESULTS: In total, 78 of the 84 students completed the study (39 students per group). The intervention resulted in a significant reduction in the mean scores of both the VAS and VMS in the experimental group, in the first and second menstrual cycles (p < 0.001, p < 0.001, respectively), but not in the means score of PBLAC. Mefenamic acid consumption at the first and second menstruation period, in the experimental group was lower than the control group (p = 0.009, p < 0.001, respectively). CONCLUSIONS: The results indicate that vitamin D supplementation could decrease the severity of primary dysmenorrhea and the need to consume pain-relief medications. Contrariwise vitamin D supplementation had no significant effect on menstrual blood loss. TRIAL REGISTRATION: This trial was registered in the Iranian Registry of Clinical Trials with code IRCT201305212324N on 18/1/2014. URL of registry: https://en.irct.ir/trial/1964 .

Association between Patients' Self-Judgement, Coagulated Menstrual Blood, and Menorrhagia: Results from a Questionnaire Survey and Blood Test Analysis.

Background and Objectives: Menorrhagia is defined as a blood loss of more than 80 mL, which is significant enough to cause anemia. Previously known methods for evaluating menorrhagia, such as the alkalin-hematin method, pictograms, and measuring the weight of sanitary products, were all impractical, complex, and time-consuming. Therefore, this study aimed to determine which item among menstrual history taking was most associated with menorrhagia and devised a simple evaluating method for menorrhagia through history taking that can be applied clinically. Materials and Methods: The study was conducted from June 2019 to December 2021. A survey was conducted on premenopausal women who underwent outpatient treatment or surgery and those who underwent a gynecologic screening test, and their blood tests were analyzed. The presence of iron deficiency anemia was identified with a Hb level of less than 10 g/dL with microcytic hypochromic anemia on a complete blood count performed within one month of the survey. A questionnaire survey was conducted on six items related to menorrhagia to investigate whether each item was related to "significant menorrhagia". Results: There were 301 participants in the survey during the period. In univariate analysis, the results revealed a statistically significant association between significant menorrhagia and the following items: self-judgement of menorrhagia; menstruation lasting over 7 days; total pad counts in a single menstrual period; Number of sanitary products changed per day; and leakaging of menstrual blood and presence of coagulated menstrual blood. In multivariate analysis, only the "self-judgement of menorrhagia" item showed a statistically significant result (p-value = 0.035; an odds ratio = 2.217). When the "self-judgement of menorrhagia" item was excluded, the "passage of clots larger than one inch in diameter" item showed a statistically significant result (p-value = 0.023; an odds ratio = 2.113). Conclusions: "Patient self-judgement of menorrhagia" is a reliable item for evaluating menorrhagia. Among several symptoms indicating menorrhagia, determining the presence of the "passage of clots larger than one inch in diameter" during the menstrual period is the most useful item for evaluating menorrhagia in clinical history taking. This study suggested using these simple menstrual history taking items to evaluate menorrhagia in real clinical practice.

Therapeutic effect of menstrual blood stem cells in rats with thin endometrium. / æœˆç»è¡€å¹²ç»†èƒžå¯¹è–„åž‹å­å®«å† è†œå¤§é¼ çš„æ²»ç–—ä¿®å¤ä½œç”¨.

OBJECTIVES: To explore the therapeutic effect of transplantation of menstrual blood stem cells (MenSCs) in rats with thin endometrium. METHODS: Thirty SPF grade female SD rats aged 8-10 weeks were randomly divided into model control group and MenSC group, with 15 rats in each group. The thin endometrium injury model was prepared by chemical method in one side of the uterus of both groups. On the 7th day of modeling, normal saline or the third generation of MenSCs were injected into the model uterus at multiple points, and the other side of the uterus was used as an internal control without treatment. HE staining was used to observe the histological structure of endometrium; immunohistochemical staining was used to observe the expression of cyto-keratin (CK) 18 and vimentin in endometrial tissue; 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay was used to evaluate the cell proliferation in endometrial tissue; immunofluorescence staining was used to detect the expression of vascular endothelial marker CD34 and vascular endothelial growth factor (VEGF) in endometrial tissue; the expression of leukemia inhibitory factor (LIF), integrin ß3 (ITGß3) and homeobox A10 (HOXA10) in endometrial tissue was determined by realtime RT-PCR. After treatments, the female and male rats were caged in a ratio of 2∶1 to observe the effect of MenSC on the reproductive function of thin endometrium model rats. RESULTS: Compared with the surgical control group, the endometrium in the model control group was thinner, and the numbers of glands and blood vessels were less (all P<0.01). After MenSC transplantation, the thickness of endometrium, the numbers of blood vessels and glands were significantly increased (all P<0.01). The proliferative cells in the basal layer of endometrium in MenSC group were more than those in the model control group (P<0.05), and the expression of vimentin, CK18, CD34 and VEGF in the uterus of rats in MenSC group were significantly higher than those in the model control group (P<0.05). LIF, ITGß3 and HOXA10 gene expression levels were also significantly higher than those in the model control group (all P<0.05). The results of pregnancy experiment showed that the number of embryo implantation in MenSC group was higher than that in model control group (P<0.05). CONCLUSIONS: MenSC transplantation can promote the proliferation of endometrial cells, upregulate vimentin, CK18, CD34 and VEGF levels, and promote the recovery of endometrial morphology and function, thus improving the endometrial receptivity and fertility of the rats with thin endometrium.

Construction of an Analysis Model of mRNA Markers in Menstrual Blood Based on Naïve Bayes and Multivariate Logistic Regression Methods.

OBJECTIVES: To establish the menstrual blood identification model based on Naïve Bayes and multivariate logistic regression methods by using specific mRNA markers in menstrual blood detection technology combined with statistical methods, and to quantitatively distinguish menstrual blood from other body fluids. METHODS: Body fluids including 86 menstrual blood, 48 peripheral blood, 48 vaginal secretions, 24 semen and 24 saliva samples were collected. RNA of the samples was extracted and cDNA was obtained by reverse transcription. Five menstrual blood-specific markers including members of the matrix metalloproteinase (MMP) family MMP3, MMP7, MMP11, progestogens associated endometrial protein (PAEP) and stanniocalcin-1 (STC1) were amplified and analyzed by electrophoresis. The results were analyzed by Naïve Bayes and multivariate logistic regression. RESULTS: The accuracy of the classification model constructed was 88.37% by Naïve Bayes and 91.86% by multivariate logistic regression. In non-menstrual blood samples, the distinguishing accuracy of peripheral blood, saliva and semen was generally higher than 90%, while the distinguishing accuracy of vaginal secretions was lower, which were 16.67% and 33.33%, respectively. CONCLUSIONS: The mRNA detection technology combined with statistical methods can be used to establish a classification and discrimination model for menstrual blood, which can distignuish the menstrual blood and other body fluids, and quantitative description of analysis results, which has a certain application value in body fluid stain identification.

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

BACKGROUND: Endometriosis 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. METHODS: We 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). RESULTS: We 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. CONCLUSIONS: We 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.

Greater angiogenic and immunoregulatory potency of bFGF and 5-aza-2'-deoxycytidine pre-treated menstrual blood stem cells in compare to bone marrow stem cells in rat model of myocardial infarction.

BACKGROUND: This study is designed to compare the menstrual blood stem cells (MenSCs) and bone marrow stem cells (BMSCs)-secreted factors with or without pre-treatment regimen using basic fibroblast growth factor (bFGF) and 5-aza-2'-deoxycytidine (5-aza) and also regenerative capacity of pre-treated MenSCs and/or BMSCs in a rat model of myocardial infarction (MI). METHODS: BMSCs and MenSCs were pre-treated with bFGF and 5-aza for 48 h and we compared the paracrine activity by western blotting. Furthermore, MI model was created and the animals were divided into sham, MI, pre-treated BMSCs, and pre-treated MenSCs groups. The stem cells were administrated via tail vain. 35 days post-MI, serum and tissue were harvested for further investigations. RESULTS: Following pre-treatment, vascular endothelium growth factor, hypoxia-inducible factor-1, stromal cell-derived factor-1, and hepatocyte growth factor were significantly increased in secretome of MenSCs in compared to BMSCs. Moreover, systemic administration of pre-treated MenSCs, leaded to improvement of cardiac function, preservation of myocardium from further subsequent injuries, promotion the angiogenesis, and reduction the level of NF-κB expression in compared to the pre-treated BMSCs. Also, pre-treated MenSCs administration significantly decreased the serum level of Interleukin 1 beta (IL-1ß) in compared to the pre-treated BMSCs and MI groups. CONCLUSIONS: bFGF and 5-aza pre-treated MenSCs offer superior cardioprotection compare to bFGF and 5-aza pre-treated BMSCs following MI.

What Do the Transcriptome and Proteome of Menstrual Blood-Derived Mesenchymal Stem Cells Tell Us about Endometriosis?

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial-mesenchymal transition (↑) and PI3K signaling via AKT to mTORC1 (↓ in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (↑ in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis.

Bleeding patterns of women with heavy menstrual bleeding or dysmenorrhoea using the levonorgestrel-releasing intrauterine system: results from a real-world observational study in Japan (J-MIRAI).

PURPOSE: To collect real-world safety and clinical outcome data on the levonorgestrel-releasing intrauterine system (LNG-IUS) for functional/organic heavy menstrual bleeding (HMB) and dysmenorrhoea in Japanese women (J-MIRAI). MATERIALS AND METHODS: In this prospective, multicentre, single-cohort, open-label, post-authorisation study, we assessed menstrual blood loss after LNG-IUS insertion by changes from baseline in pictorial blood loss assessment chart (PBAC) scores. Scores for the menorrhagia multi-attribute scale (MMAS) were collected for 12 months to assess quality of life. RESULTS: We included 47 patients with complete PBAC score and patient diary data. The median PBAC score before LNG-IUS insertion was 159.0, which decreased significantly to 6.0 at 12 months post-insertion; for patients with adenomyosis (n = 20), PBAC score decreased from 174.5 pre-insertion to 19.5 at 12 months. The number of patient-reported bleeding days was correlated with PBAC score ≥5. The proportion of women with prolonged bleeding decreased from 85.7% to 34.6% by the study's end. Some women reported no bleeding after the first 90-day reference period. The mean MMAS overall score significantly increased from 50.50 before insertion to 88.67 at 12 months. CONCLUSIONS: Japanese women with functional/organic HMB experienced substantial reductions in bleeding symptoms and improvements in quality of life after 12-month use of the LNG-IUS.