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.

Expression of vascular endothelial growth factor A isoforms is dysregulated in women with endometriosis.

Angiogenesis is a critical step in the development of ectopic lesions during endometriosis. Although total vascular endothelial growth factor (VEGF) A is elevated in the peritoneal fluid of women with endometriosis, there are contradictory reports on how levels of total endometrial VEGFA are altered in this disease. Furthermore, limited research is available on different VEGFA isoforms in women with endometriosis. Thus, the aim of the present study was to analyse levels of various VEGFA isoforms in women with and without endometriosis at different stages of the menstrual cycle. Quantitative polymerase chain reaction analysis showed that total VEGFA was highest during menstruation in endometriosis compared with controls (P=0.0373). VEGF121 and VEGF189 were similarly highest during menstruation in endometriosis compared with controls (P=0.0165 and 0.0154 respectively). The present study is also the first to identify the natural expression of VEGF111 in human tissue, which is also highest during menstruation in endometriosis (P=0.0464). This discovery of the natural production of VEGF111 in human endometrium, as well as the upregulation of VEGFA isoforms during menstruation in endometriosis, may shed further light on the development and progression of the disease, and improve our understanding of the regulation of endometrial angiogenesis.

Identification of genes differentially expressed in menstrual breakdown and repair.

STUDY QUESTION: Does the changing molecular profile of the endometrium during menstruation correlate with the histological profile of menstruation. SUMMARY ANSWER: We identified several genes not previously associated with menstruation; on Day 2 of menstruation (early-menstruation), processes related to inflammation are predominantly up-regulated and on Day 4 (late-menstruation), the endometrium is predominantly repairing and regenerating. WHAT IS KNOWN ALREADY: Menstruation is induced by progesterone withdrawal at the end of the menstrual cycle and involves endometrial tissue breakdown, regeneration and repair. Perturbations in the regulation of menstruation may result in menstrual disorders including abnormal uterine bleeding. STUDY DESIGN, SIZE DURATION: Endometrial samples were collected by Pipelle biopsy on Days 2 (n = 9), 3 (n = 9) or 4 (n = 6) of menstruation. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA was extracted from endometrial biopsies and analysed by genome wide expression Illumina Sentrix Human HT12 arrays. Data were analysed using 'Remove Unwanted Variation-inverse (RUV-inv)'. Ingenuity pathway analysis (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 were used to identify canonical pathways, upstream regulators and functional gene clusters enriched between Days 2, 3 and 4 of menstruation. Selected individual genes were validated by quantitative PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 1753 genes were differentially expressed in one or more comparisons. Significant canonical pathways, gene clusters and upstream regulators enriched during menstrual bleeding included those associated with immune cell trafficking, inflammation, cell cycle regulation, extracellular remodelling and the complement and coagulation cascade. We provide the first evidence for a role for glutathione-mediated detoxification (glutathione-S-transferase mu 1 and 2; GSTM1 and GSTM2) during menstruation. The largest number of differentially expressed genes was between Days 2 and 4 of menstruation (n = 1176). We identified several genes not previously associated with menstruation including lipopolysaccharide binding protein, serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) and -4 (SERPINB4), interleukin-17C (IL17C), V-set domain containing T-cell activation inhibitor 1 (VTCN1), proliferating cell nuclear antigen factor (KIAA0101/PAF), trefoil factor 3 (TFF3), laminin alpha 2 (LAMA2) and serine peptidase inhibitor, Kazal type 1 (SPINK1). Genes related to inflammatory processes were up-regulated on Day 2 (early-menstruation), and those associated with endometrial repair and regeneration were up-regulated on Day 4 (late-menstruation). LIMITATIONS, REASONS FOR CAUTION: Participants presented with a variety of endometrial pathologies related to bleeding status and other menstrual characteristics. These variations may also have influenced the menstrual process. WIDER IMPLICATIONS OF THE FINDINGS: The temporal molecular profile of menstruation presented in this study identifies a number of genes not previously associated with the menstrual process. Our findings provide valuable insight into the menstrual process and may present novel targets for therapeutic intervention in cases of endometrial dysfunction. LARGE SCALE DATA: All microarray data have been deposited in the public data repository Gene Expression Omnibus (GSE86003). STUDY FUNDING AND COMPETING INTERESTS: Funding for this work was provided by a National Health and Medical Research Council of Australia (NHMRC) Project Grant APP1008553 to M.H., P.R. and J.G. M.H. is supported by an NHMRC Practitioner Fellowship. P.P. is supported by a NHMRC Early Career Fellowship. The authors have no conflict of interest to declare.

TMPRSS6 rs855791 polymorphism influences the susceptibility to iron deficiency anemia in women at reproductive age.

BACKGROUND: Genome-wide-association studies have identified the TMPRSS6 polymorphism rs855791 has the strongest association with red blood cell indices or iron parameters in general population. Whether this genetic variant influences the susceptibility of iron deficiency anemia (IDA) in women with menstruation has not been well studied. METHODS: In this case-control study, we enrolled 67 women with IDA and 107 healthy volunteers, and analyzed their complete blood counts, rs855791 genotypes, and menstrual amounts. Menstrual blood loss was evaluated with a pictorial blood-loss assessment chart. RESULTS: There were significantly fewer rs855791 C homozygotes in the IDA group than in the healthy group (11.9% vs. 25.2%, p = 0.03). The odds ratio (OR) of C homozygotes having IDA versus non-CC subjects having IDA was 0.4 (95% CI, 0.17 - 0.95, p = 0.04). When the analysis was confined to study subjects with menorrhagia, this difference became more prominent (9.6% vs. 28.6%, p = 0.01; OR, 0.27, 95% CI, 0.09 - 0.77, p = 0.01). For women with non-CC genotypes, there was an inverse correlation between hemoglobin levels and menstrual loss (p < 0.001); however, this association was not found for those with genotypes CC (p = 0.15). CONCLUSIONS: Our study suggests homozygosity for TMPRSS6 rs855791 C genotype has a protective role against IDA in women at reproductive age, especially in those with menorrhagia.

Influence of diet, menstruation and genetic factors on iron status: a cross-sectional study in Spanish women of childbearing age.

The aim of this study was to investigate the combined influence of diet, menstruation and genetic factors on iron status in Spanish menstruating women (n = 142). Dietary intake was assessed by a 72-h detailed dietary report and menstrual blood loss by a questionnaire, to determine a Menstrual Blood Loss Coefficient (MBLC). Five selected SNPs were genotyped: rs3811647, rs1799852 (Tf gene); rs1375515 (CACNA2D3 gene); and rs1800562 and rs1799945 (HFE gene, mutations C282Y and H63D, respectively). Iron biomarkers were determined and cluster analysis was performed. Differences among clusters in dietary intake, menstrual blood loss parameters and genotype frequencies distribution were studied. A categorical regression was performed to identify factors associated with cluster belonging. Three clusters were identified: women with poor iron status close to developing iron deficiency anemia (Cluster 1, n = 26); women with mild iron deficiency (Cluster 2, n = 59) and women with normal iron status (Cluster 3, n = 57). Three independent factors, red meat consumption, MBLC and mutation C282Y, were included in the model that better explained cluster belonging (R2 = 0.142, p < 0.001). In conclusion, the combination of high red meat consumption, low menstrual blood loss and the HFE C282Y mutation may protect from iron deficiency in women of childbearing age. These findings could be useful to implement adequate strategies to prevent iron deficiency anemia.

Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells.

Choosing appropriate reference genes or internal controls to normalize RT-qPCR data is mandatory for the interexperimental reproducibility of gene expression data obtained by RT-qPCR in most studies, including those on endometriosis. Particularly for miRNAs, the choice for reference genes is challenging because of their physicochemical and biological characteristics. Moreover, the retrograde menstruation theory, mesenchymal stem cells in menstrual blood (MenSCs), and changes in post-transcriptional regulatory processes through miRNAs have gained prominence in the scientific community as important players in endometriosis. Therefore, we originally explored the stability of 10 miRNAs expressions as internal control candidates in conditions involving the two-dimensional culture of MenSCs from healthy women and patients with endometriosis. Here, we applied multiple algorithms (geNorm, NormFinder, Bestkeeper, and delta Ct) to screen reference genes and assessed the comprehensive stability classification of miRNAs using RefFinder. Pairwise variation calculated using geNorm identified three miRNAs as a sufficient number of reference genes for accurate normalization. MiR-191-5p, miR-24-3p, and miR-103a-3p were the best combination for suitable gene expression normalization. This study will benefit similar research, but is also attractive for regenerative medicine and clinics that use MenSCs, miRNA expression, and RT-qPCR.

Genomic expression patterns in menstrual-related migraine in adolescents.

BACKGROUND: Exacerbation of migraine with menses is common in adolescent girls and women with migraine, occurring in up to 60% of females with migraine. These migraines are oftentimes longer and more disabling and may be related to estrogen levels and hormonal fluctuations. OBJECTIVE: This study identifies the unique genomic expression pattern of menstrual-related migraine (MRM) in comparison to migraine occurring outside the menstrual period and headache-free controls. METHODS: Whole blood samples were obtained from female subjects having an acute migraine during their menstrual period (MRM) or outside of their menstrual period (non-MRM) and controls (C)--females having a menstrual period without any history of headache. The messenger RNA was isolated from these samples, and genomic profile was assessed. Affymetrix Human Exon ST 1.0 (Affymetrix, Santa Clara, CA, USA) arrays were used to examine the genomic expression pattern differences between these 3 groups. RESULTS: Blood genomic expression patterns were obtained on 56 subjects (MRM = 18, non-MRM = 18, and controls = 20). Unique genomic expression patterns were observed for both MRM and non-MRM. For MRM, 77 genes were identified that were unique to MRM, while 61 genes were commonly expressed for MRM and non-MRM, and 127 genes appeared to have a unique expression pattern for non-MRM. In addition, there were 279 genes that differentially expressed for MRM compared to non-MRM that were not differentially expressed for non-MRM. Gene ontology of these samples indicated many of these groups of genes were functionally related and included categories of immunomodulation/inflammation, mitochondrial function, and DNA homeostasis. CONCLUSIONS: Blood genomic patterns can accurately differentiate MRM from non-MRM. These results indicate that MRM involves a unique molecular biology pathway that can be identified with a specific biomarker and suggest that individuals with MRM have a different underlying genetic etiology.

[A new method of identifying the peripheral blood and the menstrual blood].

OBJECTIVE: To explore the tissue-specific gene expressions of the peripheral blood and the menstrual blood, and to search some specific factors to establish an effective method for identifying the peripheral blood and the menstrual blood. METHODS: The specific products of the peripheral blood and the menstrual blood were detected by RT-PCR and separated by electrophoretic technology. RESULTS: Beta-spectrin (SPTB) as one specific marker of peripheral blood and 18S rRNA as a kind of the housekeeping gene were expressed in both the peripheral blood and the menstrual blood. However, matrix metalloproteinase 7 (MMP7) as one specific marker of menstrual blood and human beta defensin 1 (HBD1) as one specific marker of vaginal discharge were only found in the menstrual blood. CONCLUSION: There are differences of specific gene expressions between the peripheral blood and the menstrual blood. They could be accurately distinguished from each other by using the combination of fluorescence technology and RT-PCR to detect the specific identification of mRNA.

Endometrial macrophages in health and disease.

Macrophages are present in the endometrium throughout the menstrual cycle and are most abundant during menstruation. Endometrial macrophages contribute to tissue remodeling during establishment of pregnancy and are thought to play key roles in mediating tissue breakdown and repair during menstruation. Despite these important roles, the phenotype and function of endometrial macrophages remains poorly understood. In this review, we summarize approaches used to characterize endometrial macrophage phenotype, current understanding of the functional role of macrophages in normal endometrial physiology as well as the putative contribution of macrophage dysfunction to women's reproductive health disorders.

Role of SIRT1 and Progesterone Resistance in Normal and Abnormal Endometrium.

CONTEXT: Progesterone resistance, a known pathologic condition associated with a reduced cellular response to progesterone and heightened estrogen responses, appears to have a normal physiologic role in mammalian reproduction. The molecular mechanism responsible for progesterone resistance in normal and abnormal endometrium remains unclear. OBJECTIVE: To examine the roles of sirtuin-1 (SIRT1) in normal endometrium as well as endometrium associated with infertility and endometriosis, as an epigenetic modulator associated with progesterone resistance. METHODS: SIRT1 expression was examined by Western blot, quantitative real-time polymerase chain reaction, and immunohistochemistry in mouse uterus and human endometrium. Mice with uterine specific Sirt1 overexpression were developed to examine SIRT1's role in endometrial function and endometriosis development. EX-527, a SIRT1 inhibitor, and SRT1720, a SIRT1 agonist, were also used to evaluate SIRT1 effect on endometriosis. RESULTS: In normal healthy women, endometrial SIRT1 is expressed only during menses. SIRT1 was dramatically overexpressed in the endometrium from women with endometriosis in both the epithelium and stroma. In mice, SIRT1 is expressed at the time of implantation between day 4.5 and 5.5 of pregnancy. Overexpression of SIRT1 in the mouse uterus leads to subfertility due to implantation failure, decidualization defects and progesterone resistance. SIRT1 overexpression in endometriotic lesions promotes worsening endometriosis development. EX-527 significantly reduced the number of endometriotic lesions in the mouse endometriosis model. CONCLUSIONS: SIRT1 expression and progesterone resistance appears to play roles in normal endometrial functions. Aberrant SIRT1 expression contributes to progesterone resistance and may participate in the pathophysiology of endometriosis. SIRT1 is a novel and targetable protein for the diagnosis as well as treatment of endometriosis and the associated infertility seen in this disease.

Total and endothelial cell-derived cell-free DNA in blood plasma does not change during menstruation.

Assays measuring cell-free DNA (cfDNA) in blood have widespread potential in modern medicine. However, a comprehensive understanding of cfDNA dynamics in healthy individuals is required to assist in the design of assays that maximise the signal driven by pathological changes, while excluding fluctuations that are part of healthy physiological processes. The menstrual cycle involves major remodelling of endometrial tissue and associated apoptosis, yet there has been little investigation of the impact of the menstrual cycle on cfDNA levels. Paired plasma samples were collected from 40 healthy women on menstruating (M) and non-menstruating (NM) days of their cycle. We measured total cfDNA by targeting ALU repetitive sequences and measured endothelial-derived cfDNA by methylation-specific qPCR targeting an endothelium-unique unmethylated CDH5 DNA region. CfDNA integrity and endothelial cfDNA concentration, but not total cfDNA, are consistent across time between NM and M. No significant changes in total (ALU-115 p = 0.273; ALU-247 p = 0.385) or endothelial cell specific (p = 0.301) cfDNA were observed, leading to the conclusion that menstrual status at the time of diagnostic blood collection should not have a significant impact on the quantitation of total cfDNA and methylation-based cancer assays.

Causal relationship between the timing of menarche and young adult body mass index with consideration to a trend of consistently decreasing age at menarche.

Younger age at menarche (AAM) is associated with higher body mass index (BMI) for young women. Considering that continuous trends in decreasing AAM and increasing BMI are found in many countries, we attempted to assess whether the observed negative association between AAM and young adult BMI is causal. We included 4,093 women from the Korean Genome and Epidemiology Study (KoGES) and Healthy twin Study (HTS) with relevant epidemiologic data and genome-wide marker information. To mitigate the remarkable differences in AAM across generations, we converted the AAM to a generation-standardized AAM (gsAAM). To test causality, we applied the Mendelian randomization (MR) approach, using a genetic risk score (GRS) based on 14 AAM-associated single nucleotide polymorphisms (SNPs). We constructed MR models adjusting for education level and validated the results using the inverse-variance weighted (IVW), weighted median (WM), MR-pleiotropy residual sum and outliers test (MR-PRESSO), and MR-Egger regression methods. We found a null association using observed AAM and BMI level (conventional regression; -0.05 [95% CIs -0.10-0.00] per 1-year higher AAM). This null association was replicated when gsAAM was applied instead of AAM. Using the two-stage least squares (2SLS) approach employing a univariate GRS, the association was also negated for both AAM and gsAAM, regardless of model specifications. All the MR diagnostics suggested statistically insignificant associations, but weakly negative trends, without evidence of confounding from pleiotropy. We did not observe a causal association between AAM and young adult BMI whether we considered the birth cohort effect or not. Our study alone does not exclude the possibility of existing a weak negative association, considering the modest power of our study design.

Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain.

BACKGROUND: Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix GeneChip rhesus Macaque Genome Array.Having shown that qRT-PCR and Affymetrix GeneChip data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages. RESULTS: Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip facilitated more targeted analysis than could be accomplished using the rhesus GeneChip. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed. CONCLUSIONS: Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.

Endocrine regulation of menstruation.

In women, endometrial morphology and function undergo characteristic changes every menstrual cycle. These changes are crucial for perpetuation of the species and are orchestrated to prepare the endometrium for implantation of a conceptus. In the absence of pregnancy, the human endometrium is sloughed off at menstruation over a period of a few days. Tissue repair, growth, angiogenesis, differentiation, and receptivity ensue to prepare the endometrium for implantation in the next cycle. Ovarian sex steroids through interaction with different cognate nuclear receptors regulate the expression of a cascade of local factors within the endometrium that act in an autocrine/paracrine and even intracrine manner. Such interactions initiate complex events within the endometrium that are crucial for implantation and, in the absence thereof, normal menstruation. A clearer understanding of regulation of normal endometrial function will provide an insight into causes of menstrual dysfunction such as menorrhagia (heavy menstrual bleeding) and dysmenorrhea (painful periods). The molecular pathways that precipitate these pathologies remain largely undefined. Future research efforts to provide greater insight into these pathways will lead to the development of novel drugs that would target identified aberrations in expression and/or of local uterine factors that are crucial for normal endometrial function.

The ecology and evolutionary endocrinology of reproduction in the human female.

Human reproductive ecology (HRE) is the study of the mechanisms that link variation in reproductive traits with variation in local habitats. Empirical and theoretical contributions from biological anthropology, physiology, and demography have established the foundation necessary for developing a comprehensive understanding, grounded in life history theory (LHT), of temporal, individual, and populational variation in women's reproductive functioning. LHT posits that natural selection leads to the evolution of mechanisms that tend to allocate resources to the competing demands of growth, reproduction, and survival such that fitness is locally maximized. (That is, among alternative allocation patterns exhibited in a population, those having the highest inclusive fitness will become more common over generational time.) Hence, strategic modulation of reproductive effort is potentially adaptive because investment in a new conception may risk one's own survival, future reproductive opportunities, and/or current offspring survival. The hypothalamic-pituitary-ovarian (HPO) axis is the principal neuroendocrine pathway by which the human female modulates reproductive functioning according to the changing conditions in her habitat. Adjustments of reproductive investment in a potential conception are manifested in temporal and individual variation in ovarian cycle length, ovulation, hormone levels, and the probability of conception. Understanding the extent and causes of adaptive and non-adaptive variation in ovarian functioning is fundamental to ascertaining the proximate and remote determinants of human reproductive patterns. In this review I consider what is known and what still needs to be learned of the ecology of women's reproductive biology, beginning with a discussion of the principal explanatory frameworks in HRE and the biometry of ovarian functioning. Turning next to empirical studies, it is evident that marked variation between cycles, women, and populations is the norm rather than an aberration. Other than woman's age, the determinants of these differences are not well characterized, although developmental conditions, dietary practices, genetic variation, and epigenetic mechanisms have all been hypothesized to play some role. It is also evident that the reproductive functioning of women born and living in arduous conditions is not analogous to that of athletes, dieters, or even the lower end of the "normal range" of HPO functioning in wealthier populations. Contrary to the presumption that humans have low fecundity and an inefficient reproductive system, both theory and present evidence suggest that we may actually have very high fecundity and a reproductive system that has evolved to be flexible, ruthlessly efficient and, most importantly, strategic.

Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

CONTEXT: Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. OBJECTIVE AND DESIGN: Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. RESULTS: IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. CONCLUSIONS: Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

Transient trimethylaminuria related to menstruation.

BACKGROUND: Trimethylaminuria, or fish odor syndrome, includes a transient or mild malodor caused by an excessive amount of malodorous trimethylamine as a result of body secretions. Herein, we describe data to support the proposal that menses can be an additional factor causing transient trimethylaminuria in self-reported subjects suffering from malodor and even in healthy women harboring functionally active flavin-containing monooxygenase 3 (FMO3). METHODS: FMO3 metabolic capacity (conversion of trimethylamine to trimethylamine N-oxide) was defined as the urinary ratio of trimethylamine N-oxide to total trimethylamine. RESULTS: Self-reported Case (A) that was homozygous for inactive Arg500stop FMO3, showed decreased metabolic capacity of FMO3 (i.e., approximately 10% the unaffected metabolic capacity) during 120 days of observation. For Case (B) that was homozygous for common [Glu158Lys; Glu308Gly] FMO3 polymorphisms, metabolic capacity of FMO3 was almost approximately 90%, except for a few days surrounding menstruation showing < 40% metabolic capacity. In comparison, three healthy control subjects that harbored heterozygous polymorphisms for [Glu158Lys; Glu308Gly] FMO3 or homozygous for wild FMO3 showed normal (> 90%) metabolic capacity, however, on days around menstruation the FMO3 metabolic capacity was decreased to ~60-70%. CONCLUSION: Together, these results indicate that abnormal FMO3 capacity is caused by menstruation particularly in the presence, in homozygous form, of mild genetic variants such as [Glu158Lys; Glu308Gly] that cause a reduced FMO3 function.

Reduced Transforming Growth Factor-ß Activity in the Endometrium of Women With Heavy Menstrual Bleeding.

Context: Heavy menstrual bleeding (HMB) is common and incapacitating. Aberrant menstrual endometrial repair may result in HMB. The transforming growth factor (TGF)-ß superfamily contributes to tissue repair, but its role in HMB is unknown. Objective: We hypothesized that TGF-ß1 is important for endometrial repair, and women with HMB have aberrant TGF-ß1 activity at menses. Participants/Setting: Endometrial biopsies were collected from women, and menstrual blood loss objectively measured [HMB >80 mL/cycle; normal menstrual bleeding (NMB) <80 mL]. Design: Immunohistochemistry and reverse transcription polymerase chain reaction examined endometrial TGF-ß1 ligand, receptors, and downstream SMADs in women with NMB and HMB. The function and regulation of TGF-ß1 were examined using cell culture. Results: TGFB1 mRNA was maximal immediately prior to menses, but no differences detected between women with NMB and HMB at any cycle stage. Histoscoring of TGFB1 revealed reduced staining in the stroma during menses in women with HMB (P < 0.05). There were no significant differences in TGFBR1/2 or TGFBR1/2 immunostaining. Cortisol increased activation of TGFB1 in the supernatant of human endometrial stromal cells (HES; P < 0.05) via thrombospondin-1. Endometrial SMAD2 and SMAD3 were lower in women with HMB during menstruation (P < 0.05), and decreased phosphorylated SMAD2/3 immunostaining was seen in glandular epithelial cells during the late secretory phase (P < 0.05). Wound scratch assays revealed increased repair in HES cells treated with TGF-ß1 versus control (P < 0.05). Conclusions: Women with HMB had decreased TGF-ß1 and SMADs perimenstrually. Cortisol activated latent TGF-ß1 to enhance endometrial stromal cell repair. Decreased TGF-ß1 activity may hinder repair of the denuded menstrual endometrium, resulting in HMB.

Investigation of polymorphisms in genes involved in estrogen metabolism in menstrual migraine.

Migraine is a common, disabling headache disorder, which is influenced by multiple genes and environmental triggers. After puberty, the prevalence of migraine in women is three times higher than in men and >50% of females suffering from migraine report a menstrual association, suggesting hormonal fluctuations can influence the risk of migraine attacks. It has been hypothesized that the drop in estrogen during menses is an important trigger for menstrual migraine. Catechol-O-methyltransferase (COMT) and Cytochrome P450 (CYP) enzymes are involved in estrogen synthesis and metabolism. Functional polymorphisms in these genes can influence estrogen levels and therefore may be associated with risk of menstrual migraine. In this study we investigated four single nucleotide polymorphisms in three genes involved in estrogen metabolism that have been reported to impact enzyme levels or function, in a specific menstrual migraine cohort. 268 menstrual migraine cases and 142 controls were genotyped for rs4680 in COMT (Val158Met), rs4646903 and rs1048943 in CYP1A1 (T3801C and Ile462Val) and rs700519 in CYP19A1 (Cys264Arg). Neither genotype nor allele frequencies for the COMT and CYP SNPs genotyped were found to be significantly different between menstrual migraineurs and controls by chi-square analysis (P>0.05). Therefore we did not find association of functional polymorphisms in the estrogen metabolism genes COMT, CYP1A1 or CYP19A1 with menstrual migraine. Further studies are required to assess whether menstrual migraine is genetically distinct from the common migraine subtypes and identify genes that influence risk.

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair.

Context: Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined. Objective: To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses. Patients/Setting: Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood. Design: CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture. Results: CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone. Conclusions: These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4.