Cyclical endometrial repair and regeneration.

Uniquely among adult tissues, the human endometrium undergoes cyclical shedding, scar-free repair and regeneration during a woman's reproductive life. Therefore, it presents an outstanding model for study of such processes. This Review examines what is known of endometrial repair and regeneration following menstruation and parturition, including comparisons with wound repair and the influence of menstrual fluid components. We also discuss the contribution of endometrial stem/progenitor cells to endometrial regeneration, including the importance of the stem cell niche and stem cell-derived extracellular vesicles. Finally, we comment on the value of endometrial epithelial organoids to extend our understanding of endometrial development and regeneration, as well as therapeutic applications.

Menstrual fluid endometrial stem/progenitor cell and supernatant protein content: cyclical variation and indicative range.

STUDY QUESTION: Does natural variation exist in the endometrial stem/progenitor cell and protein composition of menstrual fluid across menstrual cycles in women? SUMMARY ANSWER: Limited variation exists in the percentage of some endometrial stem/progenitor cell types and abundance of selected proteins in menstrual fluid within and between a cohort of women. WHAT IS KNOWN ALREADY: Menstrual fluid is a readily available biofluid that can represent the endometrial environment, containing endometrial stem/progenitor cells and protein factors. It is unknown whether there is natural variation in the cellular and protein content across menstrual cycles of individual women, which has significant implications for the use of menstrual fluid in research and clinical applications. STUDY DESIGN, SIZE, DURATION: Menstrual fluid was collected from 11 non-pregnant females with regular menstrual cycles. Participants had not used hormonal medications in the previous 3 months. Participants collected menstrual fluid samples from up to five cycles using a silicone menstrual cup worn on Day 2 of menstrual bleeding. PARTICIPANTS/MATERIALS, SETTING, METHODS: Menstrual fluid samples were centrifuged to separate soluble proteins and cells. Cells were depleted of red blood cells and CD45+ leucocytes. Menstrual fluid-derived endometrial stem/progenitor cells were characterized using multicolour flow cytometry including markers for endometrial stem/progenitor cells N-cadherin (NCAD) and stage-specific embryonic antigen-1 (SSEA-1) (for endometrial epithelial progenitor cells; eEPC), and sushi domain containing-2 (SUSD2) (for endometrial mesenchymal stem cells; eMSC). The clonogenicity of menstrual fluid-derived endometrial cells was assessed using colony forming unit assays. Menstrual fluid supernatant was analyzed using a custom magnetic Luminex assay. MAIN RESULTS AND THE ROLE OF CHANCE: Endometrial stem/progenitor cells are shed in menstrual fluid and demonstrate clonogenic properties. The intraparticipant agreement for SUSD2+ menstrual fluid-derived eMSC (MF-eMSC), SSEA-1+ and NCAD+SSEA-1+ MF-eEPC, and stromal clonogenicity were moderate-good (intraclass correlation; ICC: 0.75, 0.56, 0.54 and 0.52, respectively), indicating limited variability across menstrual cycles. Endometrial inflammatory and repair proteins were detectable in menstrual fluid supernatant, with five of eight (63%) factors demonstrating moderate intraparticipant agreement (secretory leukocyte protein inhibitor (SLPI), lipocalin-2 (NGAL), lactoferrin, follistatin-like 1 (FSTL1), human epididymis protein-4 (HE4); ICC ranges: 0.57-0.69). Interparticipant variation was limited for healthy participants, with the exception of key outliers of which some had self-reported menstrual pathologies. LARGE SCALE DATA: N/A. There are no OMICS or other data sets relevant to this study. LIMITATIONS, REASONS FOR CAUTION: The main limitations to this research relate to the difficulty of obtaining menstrual fluid samples across multiple menstrual cycles in a consistent manner. Several participants could only donate across <3 cycles and the duration of wearing the menstrual cup varied between 4 and 6 h within and between women. Due to the limited sample size used in this study, wider studies involving multiple consecutive menstrual cycles and a larger cohort of women will be required to fully determine the normal range of endometrial stem/progenitor cell and supernatant protein content of menstrual fluid. Possibility for selection bias and true representation of the population of women should also be considered. WIDER IMPLICATIONS OF THE FINDINGS: Menstrual fluid is a reliable source of endometrial stem/progenitor cells and related endometrial proteins with diagnostic potential. The present study indicates that a single menstrual sample may be sufficient in characterizing a variety of cellular and protein parameters across women's menstrual cycles. The results also demonstrate the potential of menstrual fluid for identifying endometrial and menstrual abnormalities in both research and clinical settings as a non-invasive method for assessing endometrial health. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Australian National Health and Medical Research Council to C.E.G. (Senior Research Fellowship 1024298 and Investigator Fellowship 1173882) and to J.E. (project grant 1047756), the Monash IVF Research Foundation to C.E.G. and the Victorian Government's Operational Infrastructure Support Program. K.A.W., M.L.D.-T., S.G.S. and J.E. declare no conflicts of interest. C.E.G. reports grants from NHMRC, during the conduct of the study; grants from EndoFound USA, grants from Ferring Research Innovation, grants from United States Department of Defence, grants from Clue-Utopia Research Foundation, outside the submitted work. CEF reports grants from EndoFound USA, grants from Clue-Utopia Research Foundation, outside the submitted work.

Hormonal oral contraceptive influence on isolation, Characterization and cryopreservation of mesenchymal stem cells from menstrual fluid.

The purpose of the study was to evaluate whether the intake of hormonal oral contraceptive influences the viability of mesenchymal stem cell. Sixteen healthy female volunteers with regular menstrual cycles were invited to participate. Menstrual fluid was collected on the day of maximum flux, and collected cells were analyzed by a 'minimal standard' for MSC characterization: plastic adherence, trilineage (adipogenic, osteogenic, chondrogenic) in vitro differentiation and a minimalistic panel of markers assessed by flow cytometry (CD731, CD901, CD1051, CD34-, CD45-) using monoclonal antibodies. The participants were divided into two groups: Group 1 - no hormonal contraceptive use; Group 2 - hormonal oral contraceptive use. The median of the menstrual fluid volume was 5.0 and the median number of cells was 5.2 × 106. Median of cell viability was 89.3%. After culture, mesenchymal stem cells increased from 0.031% of the total cells to 96.9%. The cells formed clusters and reached confluence after 15-21 days of culture in the first passage. In the second passage, clusters and the confluence were observed after 3 days of culture. No difference was observed between the groups. Our data suggest that oral hormonal contraceptive intake maintains the viability of mesenchymal stem cells from menstrual fluid.

Normality Ranges of Menstrual Fluid Volume During Reproductive Life Using Direct Quantification of Menses with Vaginal Cups.

OBJECTIVE: To determine the normal limits of menstrual fluid volume during reproductive life, quantified by direct measurement. METHODS: This was an observational, prospective clinical trial of healthy women aged 20-49 years old, with normal menstrual periods, recruited in a Natural Family Planning Unit. Women collected their menstrual fluid for at least 3 menstrual periods using a vaginal cup. Menstrual volume and different covariables were evaluated using a multilevel mixed-effects linear regression. RESULTS: Ninety-six cycles from 28 patients between 24 and 49 years old were analyzed. The average menstrual volume was 86.7 mL with a range from 15 to 271 mL. The 50th percentile of all samples was 81 mL and the 95th percentile was 162 mL. For multiparous patients the 50th percentile was 93 mL and the 95th was 169 mL. Menstrual fluid volume was higher in multigravida (99.1 mL) than in nulliparous women (45.9 Ml; p < 0.02). No statistically significant associations were identified between different variables and menstrual volume. CONCLUSION: A menstrual volume over 169 mL should be considered abnormal on multiparous patients. Age was not associated with changes on menstrual fluid volume.

Marker evaluation for differentiation of blood and menstrual fluid by methylation-sensitive SNaPshot analysis.

The differentiation of blood and menstrual fluid is especially important in cases of alleged sexual assault. While the identification of blood is relatively straightforward, the identification of menstrual fluid in trace evidence has been shown to be more challenging. This may be due to the complex nature of menstrual fluid that leads to intra- and inter-individual differences in composition. Nevertheless, recent advances in DNA methylation profiling have revealed promising markers for the differentiation of the two body fluids and furthermore, markers to distinguish menstrual fluid from vaginal fluid. A literature study was performed and in total, 11 markers were evaluated in this study of which seven could be validated for menstrual fluid and blood identification purposes. Marker "BLU2" (chr16:29757334) was identified as most suitable for differentiation of blood and menstrual fluid.

Novel messenger RNAs for body fluid identification.

In forensic investigations, the identification of the cellular or body fluid source of biological evidence can provide crucial probative information for the court. Messenger RNA (mRNA) profiling has become a valuable tool for body fluid and cell type identification due to its high sensitivity and compatibility with DNA analysis. However, using a single marker to determine the somatic origin of a sample can lead to misinterpretation as a result of cross-reactions. While false positives may be avoided through the simultaneous detection of multiple markers per body fluid, this approach is currently limited by the small number of known differentially expressed mRNAs. Here we characterise six novel mRNAs, partly identified from RNA-Seq, which can supplement existing markers for the detection of circulatory blood, semen (with and without spermatozoa), and menstrual fluid: HBD and SLC4A1 for blood, TNP1 for spermatozoa, KLK2 for seminal fluid, and MMP3 and STC1 for menstrual fluid. Respective expression profiles were evaluated by singleplex endpoint reverse transcription polymerase chain reaction (RT-PCR). HBD, SLC4A1, and KLK2 were specific to their target body fluids. TNP1, MMP3, and STC1 each cross-reacted with two non-target samples; however, these signals were below 350RFU, not reproducible, and likely resulted from large body fluid inputs. All candidates were more sensitive for the detection of their target body fluids than corresponding well-known mRNAs, in particular those for menstrual fluid. The increased sensitivities were statistically significant, except for KLK2. Thus, the new mRNAs introduced here are promising new targets for improved body fluid profiling.

Investigating Menstruation and Adverse Pregnancy Outcomes: Oxymoron or New Frontier? A Narrative Review.

Not discounting the important foetal or placental contribution, the endometrium is a key determinant of pregnancy outcomes. Given the inherently linked processes of menstruation, pregnancy and parturition with the endometrium, further understanding of menstruation will help to elucidate the maternal contribution to pregnancy. Endometrial health can be assessed via menstrual history and menstrual fluid, a cyclically shed, easily and non-invasively accessible biological sample that represents the distinct, heterogeneous composition of the endometrial environment. Menstrual fluid has been applied to the study of endometriosis, unexplained infertility and early pregnancy loss; however, it is yet to be examined regarding adverse pregnancy outcomes. These adverse outcomes, including preeclampsia, foetal growth restriction (FGR), spontaneous preterm birth and perinatal death (stillbirth and neonatal death), lay on a spectrum of severity and are often attributed to placental dysfunction. The source of this placental dysfunction is largely unknown and may be due to underlying endometrial abnormalities or endometrial interactions during placentation. We present existing evidence for the endometrial contribution to adverse pregnancy outcomes and propose that a more comprehensive understanding of menstruation can provide insight into the endometrial environment, offering great potential value as a diagnostic tool to assess pregnancy risk. As yet, this concept has hardly been explored.

From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid.

Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro. Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination.

Targeted Proteomics Analysis of Staphylococcal Superantigenic Toxins in Menstrual Fluid from Women with Menstrual Toxic Shock Syndrome (mTSS).

Menstrual toxic shock syndrome (mTSS) is a rare life-threatening febrile illness that occurs in women using intravaginal menstrual protection. It is caused by toxic shock syndrome toxin 1 (TSST-1) produced by Staphylococcus aureus, triggering a sudden onset of rash and hypotension, subsequently leading to multiple organ failure. Detecting TSST-1 and S. aureus virulence factors in menstrual fluid could accelerate the diagnosis and improve therapeutic management of mTSS. However, menstrual fluid is a highly complex matrix, making detection of bacterial toxins challenging. Here, we present a mass-spectrometry-based proteomics workflow for the targeted, quantitative analysis of four S. aureus superantigenic toxins in menstrual fluids (TSST-1, SEA, SEC, and SED). This method was applied to characterize toxin levels in menstrual fluids collected from patients with mTSS and healthy women. Toxins were detectable in samples from patients with mTSS and one healthy donor at concentrations ranging from 0 to 0.46 µg/mL for TSST-1, and 0 to 1.07 µg/mL for SEC. SEA and SED were never detected in clinical specimens, even though many S. aureus strains were positive for the corresponding genes. The method presented here could be used to explore toxin production in vivo in users of intravaginal devices to improve the diagnosis, understanding, and prevention of mTSS.

Menstrual Fluid Factors Mediate Endometrial Repair.

Menstruation is a process whereby the outer functionalis layer of the endometrium is shed each month in response to falling progesterone and estrogen levels in a non-conception cycle. Simultaneously with the tissue breakdown, the surface is re-epithelialized, protecting the wound from infection. Once menstruation is complete and estrogen levels start to rise, regeneration progresses throughout the proliferative phase of the cycle, to fully restore endometrial thickness. Endometrial repair is unique compared to tissue repair elsewhere in the adult, in that it is rapid, scar-free and occurs around 400 times during each modern woman's reproductive life. The shedding tissue and that undergoing repair is bathed in menstrual fluid, which contains live cells, cellular debris, fragments of extracellular matrix, activated leukocytes and their products, soluble cellular components and extracellular vesicles. Proteomic and other analyses have revealed some detail of these components. Menstrual fluid, along with a number of individual proteins enhances epithelial cell migration to cover the wound. This is shown in endometrial epithelial and keratinocyte cell culture models, in an ex vivo decellularized skin model and in pig wounds in vivo. Thus, the microenvironment provided by menstrual fluid, is likely responsible for the unique rapid and scar-free repair of this remarkable tissue. Insight gained from analysis of this fluid is likely to be of value not only for treating endometrial bleeding problems but also in providing potential new therapies for poorly repairing wounds such as those seen in the aged and in diabetics.

Comparison of Organoids from Menstrual Fluid and Hormone-Treated Endometrium: Novel Tools for Gynecological Research.

Endometrial organoids (EMO) are an important tool for gynecological research but have been limited by generation from (1) invasively acquired tissues and thus advanced disease states and (2) from women who are not taking hormones, thus excluding 50% of the female reproductive-aged population. We sought to overcome these limitations by generating organoids from (1) menstrual fluid (MF; MFO) using a method that enables the concurrent isolation of menstrual fluid supernatant, stromal cells, and leukocytes and (2) from biopsies and hysterectomy samples from women taking hormonal medication (EMO-H). MF was collected in a menstrual cup for 4-6 h on day 2 of menstruation. Biopsies and hysterectomies were obtained during laparoscopic surgery. Organoids were generated from all sample types, with MFO and EMO-H showing similar cell proliferation rates, proportion and localization of the endometrial basalis epithelial marker, Stage Specific Embryonic Antigen-1 (SSEA-1), and gene expression profiles. Organoids from different disease states showed the moderate clustering of epithelial secretory and androgen receptor signaling genes. Thus, MFO and EMO-H are novel organoids that share similar features to EMO but with the advantage of (1) MFO being obtained non-invasively and (2) EMO-H being obtained from 50% of the women who are not currently being studied through standard methods. Thus, MFO and EMO-H are likely to prove to be invaluable tools for gynecological research, enabling the population-wide assessment of endometrial health and personalized medicine.

Detection of five specified menstrual fluid mRNA markers over the uterine cycle.

Matrix metallopeptidases (MMPs) 7, 10, and 11 are currently the most commonly employed messenger RNAs (mRNAs) for the identification of menstrual fluid (MF) in forensic analysis. However, no comprehensive study has been carried out to date to explore their time-dependent detection in vaginal samples. This research investigated the detection of MMPs 7, 10, and 11, as well as MMP3 and stanniocalcin 1 (STC1) over the uterine cycle. The aim was to associate relative transcript levels with cycle stages and thus determine which of these transcripts is most suitable for MF identification in a forensic context. Additionally, the effect of hormonal contraceptives (HCs) on their abundance was explored. A total of 300 vaginal swab samples were collected from eight female donors, including a pregnant woman, naturally cycling women, and contraceptive users. Differences among individuals were observed, but these were not consistent within the groups. Only MMP10 and STC1 mRNA abundance appeared to be unaffected by the use of HCs. MMP3, MMP7, and MMP11 transcripts were less abundant in MF samples of some HC users. Overall, MMP3 was most specific to MF, although this transcript was still detected in one of four vaginal material (VM) samples. STC1 was less specific than MMP3 (detected in 39.6 % of VM samples). However, over the days of menstruation, STC1 was more consistently detectable than the MMPs. MMP10 was least specific, with a 78.3 % detection rate in VM samples, but the presence/absence in VM was individual-specific and consistent. MMP10 may therefore be more useful as a VM marker with elevated abundance during menstruation in some individuals. MMP7 and MMP11 were the least reliable mRNAs for MF identification, despite an increased specificity compared to MMP10. Detection rates in MF were lower than those of MMP3 and STC1, whereas detection rates in VM were higher. MMP7 abundance additionally increased approximately 2-5 days after the end of menstruation in all donors except one naturally cycling individual. In view of these results, MMP3 and STC1 were identified as the most useful MF markers for forensic use. Nevertheless, mRNA typing results need to be interpreted with utmost caution.

Comparing the Effect of TGF-ß Receptor Inhibition on Human Perivascular Mesenchymal Stromal Cells Derived from Endometrium, Bone Marrow and Adipose Tissues.

Rare perivascular mesenchymal stromal cells (MSCs) with therapeutic properties have been identified in many tissues. Their rarity necessitates extensive in vitro expansion, resulting in spontaneous differentiation, cellular senescence and apoptosis, producing therapeutic products with variable quality and decreased potency. We previously demonstrated that A83-01, a transforming growth factor beta (TGF-ß) receptor inhibitor, maintained clonogenicity and promoted the potency of culture-expanded premenopausal endometrial MSCs using functional assays and whole-transcriptome sequencing. Here, we compared the effects of A83-01 on MSCs derived from postmenopausal endometrium, menstrual blood, placenta decidua-basalis, bone marrow and adipose tissue. Sushi-domain-containing-2 (SUSD2+) and CD34+CD31-CD45- MSCs were isolated. Expanded MSCs were cultured with or without A83-01 for 7 days and assessed for MSC properties. SUSD2 identified perivascular cells in the placental decidua-basalis, and their maternal origin was validated. A83-01 promoted MSC proliferation from all sources except bone marrow and only increased SUSD2 expression and prevented apoptosis in MSCs from endometrial-derived tissues. A83-01 only improved the cloning efficiency of postmenopausal endometrial MSCs (eMSCs), and expanded adipose tissue MSCs (adMSCs) underwent significant senescence, which was mitigated by A83-01. MSCs derived from bone marrow (bmMSCs) were highly apoptotic, but A83-01 was without effect. A83-01 maintained the function and phenotype in MSCs cultured from endometrial, but not other, tissues. Our results also demonstrated that cellular SUSD2 expression directly correlates with the functional phenotype.

Prostaglandins E2 and F2α levels in human menstrual fluid by online Solid Phase Extraction coupled to Liquid Chromatography tandem Mass Spectrometry (SPE-LC-MS/MS).

This paper reports an online SPE-LC-MS/MS method for the simultaneous quantification of prostaglandins (PGE2 and PGF2α) in menstrual fluid samples. To meet this goal human peripheral serum was used as surrogate matrix. The analytes were trapped on an OASIS HLB cartridge for 3 min, for sample cleanup and enrichment, and then transferred during only 42 s to an HSS T3 C18 analytical column, for separation and analysis. Prostaglandins (PGs) were detected by selected reaction monitoring in negative ion mode, PGE2 (m/z 351 → 315) and PGF2α (m/z 353 → 193) using isotope-labeled internal standard (PGE2-d4, m/z 355 → 319). The concentration linear range was of 10.34-1.034 ng mL-1 and the lower limit of quantification (LLOQ) was 10.34 ng mL-1 for both PGs. Validation parameters were successfully assessed according to the European Medicines Agency guideline (EMA), also comprising the FDA normative. The method showed no matrix effect and process efficiency around 100%, in addition to only 15 min of analysis time with lower solvent consumption. The method application was carried out using two menstrual fluid sample groups: control (n = 15) and treatment group (n = 7; samples from women that used Tahiti lemon juice). The PGF2α levels were found to be higher in treated group than in control group (p ≤ 0.05), denoting an effect of the intake of Tahiti lemon juice on the menstrual inflammatory process. The on-line method herein reported could be useful for the analysis of PGs from large research studies.

Adhesive potential of the shed endometrial cells in menstrual fluid using amniotic membrane: establishment of in-vitro model for pathogenesis of endometriosis / 대한산부인과학회잡지

OBJECTIVES: The purpose of this study was to determine whether or not the shed endometrial tissues in menstrual fluid (MF) have adhesive potentials, using human amniotic membrane (AM). METHODS: The MF from 20 patients with regular menstruation was collected with Wallace catheter by aspiration from the uterine cavity on the second or third day of the menstrual period. The AM was obtained from the placenta of term delivery without any complication. The MF was washed and diluted fivefold with Hams F-10 medium supplemented with 10% fetal bovine serum. The cell suspension was placed on either epithelial layer (EP) or extracellular matrix layer (ECM) of the AM. After 5 days of culture, the adhesion sites were observed under a stereomicroscope. For histological observation, each cultured AM was prepared for the serial paraffin section. RESULTS: The adhesion sites of endometrial tissues in MF were found both ECM (20/20) and EP (11/20) of the AM. The size of adhesion sites in each AM were highly variable from microscopic to macroscopic size. CONCLUSION: We found that the shed endometrial tissues in MF have adhesive potential to epithelial layer in addition to extracellular matrix layer of amniotic membrane. This adhesive potential may be related to pathogenesis of endometriosis. We suggest that this culture system can be useful as an in-vitro model for endometriosis.