The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

Endometrial Proliferative Phase-Centered View of Transcriptome Dynamics across the Menstrual Cycle.

The endometrium, the inner mucosal lining of the uterus, undergoes complex molecular and cellular changes across the menstrual cycle in preparation for embryo implantation. Transcriptome-wide analyses have mainly been utilized to study endometrial receptivity, the prerequisite for successful implantation, with most studies, so far, comparing the endometrial transcriptomes between (i) secretory and proliferative endometrium or (ii) mid-secretory and early secretory endometrium. In the current study, we provide a complete transcriptome description of the endometrium across the entire menstrual cycle and, for the first time, comprehensively characterize the proliferative phase of the endometrium. Our temporal transcriptome analysis includes five time points including the mid-proliferative, late proliferative (peri-ovulatory phase), early secretory, mid-secretory, and late secretory phases. Thus, we unveil exhaustively the transitions between the consecutive proliferative and secretory phases, highlighting their unique gene expression profiles and possible distinct biological functions. The transcriptome analysis reveals many differentially expressed genes (DEGs) across the menstrual cycle, most of which are phase-specific. As an example of coordinated gene activity, the expression profile of histone-encoding genes within the HIST cluster on chromosome 6 shows an increase in cluster activity during the late proliferative and a decline during the mid-secretory phase. Moreover, numerous DEGs are shared among all phases. In conclusion, in the current study, we delineate the endometrial proliferative phase-centered view of transcriptome dynamics across the menstrual cycle. Our data analysis highlights significant transcriptomic and functional changes occurring during the late proliferative phase-an essential transition point from the proliferative phase to the secretory phase. Future studies should explore how the biology of the late proliferative phase endometrium impacts the achievement of mid-secretory endometrial receptivity or contributes to molecular aberrations leading to embryo implantation failure.

Inhibition of epigenetic and cell cycle-related targets in glioblastoma cell lines reveals that onametostat reduces proliferation and viability in both normoxic and hypoxic conditions.

The choice of targeted therapies for treatment of glioblastoma patients is currently limited, and most glioblastoma patients die from the disease recurrence. Thus, systematic studies in simplified model systems are required to pinpoint the choice of targets for further exploration in clinical settings. Here, we report screening of 5 compounds targeting epigenetic writers or erasers and 6 compounds targeting cell cycle-regulating protein kinases against 3 glioblastoma cell lines following incubation under normoxic or hypoxic conditions. The viability/proliferation assay indicated that PRMT5 inhibitor onametostat was endowed with high potency under both normoxic and hypoxic conditions in cell lines that are strongly MGMT-positive (T98-G), weakly MGMT-positive (U-251 MG), or MGMT-negative (U-87 MG). In U-251 MG and U-87 MG cells, onametostat also affected the spheroid formation at concentrations lower than the currently used chemotherapeutic drug lomustine. In T98-G cell line, treatment with onametostat led to dramatic changes in the transcriptome profile by inducing the cell cycle arrest, suppressing RNA splicing, and down-regulating several major glioblastoma cell survival pathways. Further validation by immunostaining in three cell lines confirmed that onametostat affects cell cycle and causes reduction in nucleolar protein levels. In this way, inhibition of epigenetic targets might represent a viable strategy for glioblastoma treatment even in the case of decreased chemo- and radiation sensitivity, although further studies in clinically more relevant models are required.

Induced Pluripotent Stem Cells as a Possible Approach for Exploring the Pathophysiology of Polycystic Ovary Syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine condition among women with pleiotropic sequelae possessing reproductive, metabolic, and psychological characteristics. Although the exact origin of PCOS is elusive, it is known to be a complex multigenic disorder with a genetic, epigenetic, and environmental background. However, the pathogenesis of PCOS, and the role of genetic variants in increasing the risk of the condition, are still unknown due to the lack of an appropriate study model. Since the debut of induced pluripotent stem cell (iPSC) technology, the ability of reprogrammed somatic cells to self-renew and their potential for multidirectional differentiation have made them excellent tools to study different disease mechanisms. Recently, researchers have succeeded in establishing human in vitro PCOS disease models utilizing iPSC lines from heterogeneous PCOS patient groups (iPSCPCOS). The current review sets out to summarize, for the first time, our current knowledge of the implications and challenges of iPSC technology in comprehending PCOS pathogenesis and tissue-specific disease mechanisms. Additionally, we suggest that the analysis of polygenic risk prediction based on genome-wide association studies (GWAS) could, theoretically, be utilized when creating iPSC lines as an additional research tool to identify women who are genetically susceptible to PCOS. Taken together, iPSCPCOS may provide a new paradigm for the exploration of PCOS tissue-specific disease mechanisms.

MBMethPred: a computational framework for the accurate classification of childhood medulloblastoma subgroups using data integration and AI-based approaches.

Childhood medulloblastoma is a malignant form of brain tumor that is widely classified into four subgroups based on molecular and genetic characteristics. Accurate classification of these subgroups is crucial for appropriate treatment, monitoring plans, and targeted therapies. However, misclassification between groups 3 and 4 is common. To address this issue, an AI-based R package called MBMethPred was developed based on DNA methylation and gene expression profiles of 763 medulloblastoma samples to classify subgroups using machine learning and neural network models. The developed prediction models achieved a classification accuracy of over 96% for subgroup classification by using 399 CpGs as prediction biomarkers. We also assessed the prognostic relevance of prediction biomarkers using survival analysis. Furthermore, we identified subgroup-specific drivers of medulloblastoma using functional enrichment analysis, Shapley values, and gene network analysis. In particular, the genes involved in the nervous system development process have the potential to separate medulloblastoma subgroups with 99% accuracy. Notably, our analysis identified 16 genes that were specifically significant for subgroup classification, including EP300, CXCR4, WNT4, ZIC4, MEIS1, SLC8A1, NFASC, ASCL2, KIF5C, SYNGAP1, SEMA4F, ROR1, DPYSL4, ARTN, RTN4RL1, and TLX2. Our findings contribute to enhanced survival outcomes for patients with medulloblastoma. Continued research and validation efforts are needed to further refine and expand the utility of our approach in other cancer types, advancing personalized medicine in pediatric oncology.

Targeted gene expression profiling for accurate endometrial receptivity testing.

Expressional profiling of the endometrium enables the personalised timing of the window of implantation (WOI). This study presents and evaluates a novel analytical pipeline based on a TAC-seq (Targeted Allele Counting by sequencing) method for endometrial dating. The expressional profiles were clustered, and differential expression analysis was performed on the model development group, using 63 endometrial biopsies spanning over proliferative (PE, n = 18), early-secretory (ESE, n = 18), mid-secretory (MSE, n = 17) and late-secretory (LSE, n = 10) endometrial phases of the natural cycle. A quantitative predictor model was trained on the development group and validated on sequenced samples from healthy women, consisting of 52 paired samples taken from ESE and MSE phases and five LSE phase samples from 31 individuals. Finally, the developed test was applied to 44 MSE phase samples from a study group of patients diagnosed with recurrent implantation failure (RIF). In validation samples (n = 57), we detected displaced WOI in 1.8% of the samples from fertile women. In the RIF study group, we detected a significantly higher proportion of the samples with shifted WOI than in the validation set of samples from fertile women, 15.9% and 1.8% (p = 0.012), respectively. The developed model was evaluated with an average cross-validation accuracy of 98.8% and an accuracy of 98.2% in the validation group. The developed beREADY screening model enables sensitive and dynamic detection of selected transcriptome biomarkers, providing a quantitative and accurate prediction of endometrial receptivity status.

Users' Concerns About Endometriosis on Social Media: Sentiment Analysis and Topic Modeling Study.

BACKGROUND: Endometriosis is a debilitating and difficult-to-diagnose gynecological disease. Owing to limited information and awareness, women often rely on social media platforms as a support system to engage in discussions regarding their disease-related concerns. OBJECTIVE: This study aimed to apply computational techniques to social media posts to identify discussion topics about endometriosis and to identify themes that require more attention from health care professionals and researchers. We also aimed to explore whether, amid the challenging nature of the disease, there are themes within the endometriosis community that gather posts with positive sentiments. METHODS: We retrospectively extracted posts from the subreddits r/Endo and r/endometriosis from January 2011 to April 2022. We analyzed 45,693 Reddit posts using sentiment analysis and topic modeling-based methods in machine learning. RESULTS: Since 2011, the number of posts and comments has increased steadily. The posts were categorized into 11 categories, and the highest number of posts were related to either asking for information (Question); sharing the experiences (Rant/Vent); or diagnosing and treating endometriosis, especially surgery (Surgery related). Sentiment analysis revealed that 92.09% (42,077/45,693) of posts were associated with negative sentiments, only 2.3% (1053/45,693) expressed positive feelings, and there were no categories with more positive than negative posts. Topic modeling revealed 27 major topics, and the most popular topics were Surgery, Questions/Advice, Diagnosis, and Pain. The Survey/Research topic, which brought together most research-related posts, was the last in terms of posts. CONCLUSIONS: Our study shows that posts on social media platforms can provide insights into the concerns of women with endometriosis symptoms. The analysis of the posts confirmed that women with endometriosis have to face negative emotions and pain daily. The large number of posts related to asking questions shows that women do not receive sufficient information from physicians and need community support to cope with the disease. Health care professionals should pay more attention to the symptoms and diagnosis of endometriosis, discuss these topics with patients to reduce their dissatisfaction with doctors, and contribute more to the overall well-being of women with endometriosis. Researchers should also become more involved in social media and share new science-based knowledge regarding endometriosis.

Endometrial receptivity in women of advanced age: an underrated factor in infertility.

BACKGROUND: Modern lifestyle has led to an increase in the age at conception. Advanced age is one of the critical risk factors for female-related infertility. It is well known that maternal age positively correlates with the deterioration of oocyte quality and chromosomal abnormalities in oocytes and embryos. The effect of age on endometrial function may be an equally important factor influencing implantation rate, pregnancy rate, and overall female fertility. However, there are only a few published studies on this topic, suggesting that this area has been under-explored. Improving our knowledge of endometrial aging from the biological (cellular, molecular, histological) and clinical perspectives would broaden our understanding of the risks of age-related female infertility. OBJECTIVE AND RATIONALE: The objective of this narrative review is to critically evaluate the existing literature on endometrial aging with a focus on synthesizing the evidence for the impact of endometrial aging on conception and pregnancy success. This would provide insights into existing gaps in the clinical application of research findings and promote the development of treatment options in this field. SEARCH METHODS: The review was prepared using PubMed (Medline) until February 2023 with the keywords such as 'endometrial aging', 'receptivity', 'decidualization', 'hormone', 'senescence', 'cellular', 'molecular', 'methylation', 'biological age', 'epigenetic', 'oocyte recipient', 'oocyte donation', 'embryo transfer', and 'pregnancy rate'. Articles in a language other than English were excluded. OUTCOMES: In the aging endometrium, alterations occur at the molecular, cellular, and histological levels suggesting that aging has a negative effect on endometrial biology and may impair endometrial receptivity. Additionally, advanced age influences cellular senescence, which plays an important role during the initial phase of implantation and is a major obstacle in the development of suitable senolytic agents for endometrial aging. Aging is also accountable for chronic conditions associated with inflammaging, which eventually can lead to increased pro-inflammation and tissue fibrosis. Furthermore, advanced age influences epigenetic regulation in the endometrium, thus altering the relation between its epigenetic and chronological age. The studies in oocyte donation cycles to determine the effect of age on endometrial receptivity with respect to the rates of implantation, clinical pregnancy, miscarriage, and live birth have revealed contradictory inferences indicating the need for future research on the mechanisms and corresponding causal effects of women's age on endometrial receptivity. WIDER IMPLICATIONS: Increasing age can be accountable for female infertility and IVF failures. Based on the complied observations and synthesized conclusions in this review, advanced age has been shown to have a negative impact on endometrial functioning. This information can provide recommendations for future research focusing on molecular mechanisms of age-related cellular senescence, cellular composition, and transcriptomic changes in relation to endometrial aging. Additionally, further prospective research is needed to explore newly emerging therapeutic options, such as the senolytic agents that can target endometrial aging without affecting decidualization. Moreover, clinical trial protocols, focusing on oocyte donation cycles, would be beneficial in understanding the direct clinical implications of endometrial aging on pregnancy outcomes.

N-glycans from serum IgG and total serum glycoproteins specific for endometriosis.

Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of endometrial tissue outside the uterine cavity. There are currently no definitive non-invasive diagnostic tools. Glycosylation is the most common posttranslational modification of proteins and altered glycosylation has been found in many diseases, including chronic inflammatory conditions and cancer. Sialylation and galactosylation on serum IgG have previously been found to be altered in endometriosis and serum sialylation changed after Zoladex (Goserelin Acetate) therapy. Using IgG and whole serum glycoproteins, we investigated N-glycosylation in two clinical cohorts of women with and without endometriosis. PNGase F-digested serum samples were fluorescently labelled and N-glycans were profiled by ultra-performance liquid chromatography. Clinical data was collected to link glycomic findings with metabolic and hormonal profiles. Total serum glycoprotein and IgG glycosylation differed in patients with endometriosis compared to control cases. The most significantly altered was glycan peak 3 from IgG, containing bisected biantennary glycans, which was decreased in the endometriosis cohorts (p = 0.0000005-0.018). In conclusion, this is the first pilot study to identify changes in N-glycans from whole serum glycoproteins associated with endometriosis. A larger validation study is now warranted and such studies should include the follow-up of surgically and pharmacologically treated patients.

An integrative analysis of endometrial steroid metabolism and transcriptome in relation to endometrial receptivity in in vitro fertilization patients.

OBJECTIVE: To study the relationship between the steroid concentration in the endometrium, in serum, and the gene expression level of steroid-metabolizing enzymes in the context of endometrial receptivity in in vitro fertilization (IVF) patients. DESIGN: Case-control study of 40 IVF patients recruited in the SCRaTCH study (NTR5342), a randomized controlled trial investigating pregnancy outcome after "endometrial scratching." Endometrial biopsies and serum were obtained from patients with a first failed IVF cycle randomized to the endometrial scratch in the midluteal phase of the natural cycle before the next fresh embryo transfer during the second IVF cycle. SETTING: University hopsital. PATIENTS: Twenty women with clinical pregnancy were compared with 20 women who did not conceive after fresh embryo transfer. Cases and controls were matched for primary vs. secondary infertility, embryo quality, and age. INTERVENTION: None. MAIN OUTCOME MEASURE(S): Steroid concentrations in endometrial tissue homogenates and serum were measured with liquid chromatography-mass spectrometry. The endometrial transcriptome was profiled by RNA-sequencing, followed by principal component analysis and differential expression analysis. False discovery rate-adjusted and log-fold change >|0.5| were selected as the threshold for differentially expressed genes. RESULT(S): Estrogen levels were comparable in both serum (n = 16) and endometrium (n = 40). Androgens and 17-hydroxyprogesterone were higher in serum than that in endometrium. Although steroid levels did not vary between pregnant and nonpregnant groups, subgroup analysis of primary women with infertility showed a significantly lower estrone concentration and estrone:androstenedione ratio in serum of the pregnant group (n = 5) compared with the nonpregnant group (n = 2). Expression of 34 out of 46 genes encoding the enzymes controlling the local steroid metabolism was detected, and estrogen receptor ß gene was differentially expressed between pregnant and nonpregnant women. When only the primary infertile group was considered, 28 genes were differentially expressed between pregnant and nonpregnant women, including HSD11B2, that catalyzes the conversion of cortisol into cortisone. CONCLUSION(S): Steroidomic and transcriptomic analyses show that steroid concentrations are regulated by the local metabolism in the endometrium. Although no differences were found in endometrial steroid concentration in the pregnant and nonpregnant IVF patients, primary women with infertility showed deviations in steroid levels and gene expression, indicating that a more homogeneous patient group is required to uncover the exact role of steroid metabolism in endometrial receptivity. CLINICAL TRIAL REGISTRATION NUMBER: The study was registered in the Dutch trial registry (www.trialregister.nl), registration number NL5193/NTR5342, available at https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6687. The date of registration is July 31, 2015. The first enrollment is on January 1, 2016.

The expression pattern of endometrial receptivity genes is desynchronized between endometrium and matched endometriomas.

RESEARCH QUESTION: Are paired samples of endometrium and ovarian endometriomas synchronous with each other throughout the menstrual cycle? DESIGN: The expression levels of 57 endometrial receptivity-associated genes were determined from matched endometrial and endometrioma samples (n=31) collected from women with endometriosis throughout the menstrual cycle. RESULTS: The expression profile of endometrial receptivity genes divided endometrial samples according to their menstrual cycle phase. Endometrioma samples grouped together irrespective of the menstrual cycle phase and formed a cluster distinct from endometrial samples. Pairwise comparison showed 21, 16, 33 and 23 differentially expressed genes (adjusted P < 0.001-0.05) between the lesions and endometria collected in the proliferative, early-secretory, mid-secretory and late-secretory menstrual cycle phases, respectively, confirming the distinct expression profiles of endometrium and endometrioma. CONCLUSIONS: No menstrual cycle synchronicity was found between matched eutopic and ectopic endometrium, suggesting that the concept of cycling endometrial tissue inside the endometrioma should be revised.

Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon.

Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.

Uterine fluid microRNAs are dysregulated in women with recurrent implantation failure.

STUDY QUESTION: Is the composition of microRNAs (miRNAs) in uterine fluid (UF) of women with recurrent implantation failure (RIF) different from that of healthy fertile women? SUMMARY ANSWER: The composition of miRNAs in UF of women with RIF is different from that of healthy fertile women and the dysregulated miRNAs are associated with impaired endometrial receptivity and embryo implantation. WHAT IS KNOWN ALREADY: It has previously been demonstrated that the miRNAs secreted from endometrial cells into the UF contribute to the achievement of endometrial receptivity. Endometrial miRNAs are dysregulated in women with RIF. STUDY DESIGN, SIZE, DURATION: In this descriptive laboratory case-control study, miRNA abundancy was compared between UF collected during implantation phase from healthy fertile women (n = 17) and women with RIF (n = 34), which was defined as three failed IVF cycles with high-quality embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recruitment of study subjects and sampling of UF were performed at two university clinics in Stockholm, Sweden and Tartu, Estonia. The study participants monitored their menstrual cycles using an LH test kit. The UF samples were collected on Day LH + 7-9 by flushing with saline. Samples were processed for small RNA sequencing and mapped for miRNAs. The differential abundance of miRNAs in UF was compared between the two groups using differential expression analysis (DESeq2). Further downstream analyses, including miRNA target gene prediction (miRTarBase), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (g:Profiler) and external validation using relevant published data, were performed on the dysregulated miRNAs. Two miRNAs were technically validated with quantitative real-time PCR (RT-PCR). MAIN RESULTS AND THE ROLE OF CHANCE: After processing of the sequencing data, there were 15 samples in the healthy fertile group and 33 samples in the RIF group. We found 61 differentially abundant UF miRNAs (34 upregulated and 27 downregulated) in RIF compared to healthy women with a false discovery rate of <0.05 and a fold change (FC) of ≤-2 or ≥2. When analyzed with published literature, we found that several of the differentially abundant miRNAs are expressed in endometrial epithelial cells and have been reported in endometrial extracellular vesicles and in association with endometrial receptivity and RIF. Their predicted target genes were further expressed both in the trophectodermal cells of blastocyst-stage embryos and endometrial mid-secretory epithelial cells, as assessed by publicly available single-cell transcriptome-sequencing studies. Pathway analysis further revealed that 25 pathways, having key roles in endometrial receptivity and implantation, were significantly enriched. Hsa-miR-486-5p (FC -20.32; P-value = 0.004) and hsa-miR-92b-3p (FC -9.72; P-value = 0.004) were successfully technically validated with RT-PCR. LARGE SCALE DATA: The data are available in Gene Expression Omnibus (GEO) at https://www.ncbi.nlm.nih.gov/geo/ with GEO accession number: GSE173289. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study with a limited number of study participants. Moreover, the identified differentially abundant miRNAs should be validated in a larger study cohort, and the predicted miRNA target genes and enriched pathways in RIF need to be confirmed and further explored in vitro. WIDER IMPLICATIONS OF THE FINDINGS: RIF is a major challenge in the current IVF setting with no diagnostic markers nor effective treatment options at hand. For the first time, total miRNAs have been extensively mapped in receptive phase UF of both healthy women with proven fertility and women diagnosed with RIF. Our observations shed further light on the molecular mechanisms behind RIF, with possible implications in future biomarker and clinical treatment studies. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by the Swedish Research Council (2017-00932), a joint grant from Region Stockholm and Karolinska Institutet (ALF Medicine 2020, FoUI-954072), Estonian Research Council (PRG1076), Horizon 2020 innovation (ERIN, EU952516) and European Commission and Enterprise Estonia (EU48695). The authors have no competing interests to declare for the current study.

Differences in microbial profile of endometrial fluid and tissue samples in women with in vitro fertilization failure are driven by Lactobacillus abundance.

INTRODUCTION: The endometrial microbiota has been linked to several gynecological disorders, including infertility. It has been shown that the microbial profile of endometrium could have a role in fertilization and pregnancy outcomes. In this study we aim to assess the microbial community of endometrial tissue (ET) and endometrial fluid (EF) samples in women receiving in vitro fertilization (IVF) treatment. We also search for possible associations between chronic endometritis (CE) and endometrial microbiota. MATERIAL AND METHODS: This was a cohort study involving 25 women aged between 28 and 42 years with both primary and secondary infertility and with at least one IVF failure. The ET and EF sample collection was carried out between September 2016 and November 2018. Each of the participants provided two types of samples-tissue and fluid samples (50 samples in total). A 16S rRNA sequencing was performed on both of the sample types for microbial profile evaluation. CE was diagnosed based on a CD138 immunohistochemistry where CE diagnosis was confirmed in the presence of one or more plasma cells. Microbial profiles of women with and without CE were compared in both sample types separately. RESULTS: We report no differences in the microbial composition and alpha diversity (pObserved  = 0.07, pShannon  = 0.65, pInverse Simpson  = 0.59) between the EF and ET samples of IVF patients. We show that the abundance of the genus Lactobacillus influences the variation in microbial beta diversity between and fluid samples (r2  = 0.34; false discovery rate [FDR] <9.9 × 10-5 ). We report that 32% (8/25) of the participants had differences in Lactobacillus dominance in the paired samples and these samples also present a different microbial diversity (pShannon  = 0.06, FDRweighted UniFrac  = 0.01). These results suggest that the microbial differences between ET and fluid samples are driven by the abundance of genus Lactobacillus. The microbiome of CE and without CE (ie non-CE) women in our sample set of IVF patients was similar. CONCLUSIONS: Our findings show that genus Lactobacillus dominance is an important factor influencing the microbial composition of ET and fluid samples.

Homing Peptide-Based Targeting of Tenascin-C and Fibronectin in Endometriosis.

The current diagnostic and therapeutic strategies for endometriosis are limited. Although endometriosis is a benign condition, some of its traits, such as increased cell invasion, migration, tissue inflammation, and angiogenesis are similar to cancer. Here we explored the application of homing peptides for precision delivery of diagnostic and therapeutic compounds to endometriotic lesions. First, we audited a panel of peptide phages for the binding to the cultured immortalized endometriotic epithelial 12Z and eutopic stromal HESC cell lines. The bacteriophages displaying PL1 peptide that engages with angiogenic extracellular matrix overexpressed in solid tumors showed the strongest binding to both cell lines. The receptors of PL1 peptide, tenascin C domain C (TNC-C) and fibronectin Extra Domain-B (Fn-EDB), were expressed in both cells. Silver nanoparticles functionalized with synthetic PL1 peptide showed specific internalization in 12Z and HESC cells. Treatment with PL1-nanoparticles loaded with the potent antimitotic drug monomethyl auristatin E decreased the viability of endometriotic cells in 2D and 3D cultures. Finally, PL1-nanoparticless bound to the cryosections of clinical peritoneal endometriotic lesions in the areas positive for TNC-C and Fn-EDB immunoreactivities and not to sections of normal endometrium. Our findings suggest potential applications for PL1-guided nanoparticles in precision diagnosis and therapy of endometriosis.

Potential innate immunity-related markers of endometrial receptivity and recurrent implantation failure (RIF).

The successful implantation of the embryo into a receptive endometrium is essential for the establishment of a viable pregnancy while recurrent implantation failure (RIF) is a real challenge in assisted reproduction. The maternal innate immune system, specifically the Toll-like receptors (TLRs), are involved in maintaining immunity in the female reproductive tract (FRT) required for fertility. In this study, we aimed to investigate the importance of innate immunity-related gene expression in the regulation of human fertility and as a prediction of potential outcome of in vitro fertilization - embryo transfer (IVF-ET), thus, we assessed the gene expression levels of TLR signalling molecules using quantitative real-time PCR between endometrial biopsies of healthy fertile women, and the patients experiencing RIF. Interestingly, our results showed that, TRIB2 and TLR9 genes were differentially expressed between the endometrial biopsies of healthy women and those with RIF. However, comparing expression levels of same genes between pre-receptive and receptive healthy endometrial biopsies showed different genes (ICAM1, NFKBIA, VCAM1, LIF, VEGFB, TLR5) had significantly altered expression, suggesting their involvement in endometrial receptivity. Thus, further investigations will enable us to better understand the role of these genes in the biology of FRT and as a possible target for the improvement of infertility treatments and/or development of non-hormonal contraception.

Cell-Penetrating Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models.

Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer.

Machine Learning Approaches to Classify Primary and Metastatic Cancers Using Tissue of Origin-Based DNA Methylation Profiles.

Metastatic cancers account for up to 90% of cancer-related deaths. The clear differentiation of metastatic cancers from primary cancers is crucial for cancer type identification and developing targeted treatment for each cancer type. DNA methylation patterns are suggested to be an intriguing target for cancer prediction and are also considered to be an important mediator for the transition to metastatic cancer. In the present study, we used 24 cancer types and 9303 methylome samples downloaded from publicly available data repositories, including The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). We constructed machine learning classifiers to discriminate metastatic, primary, and non-cancerous methylome samples. We applied support vector machines (SVM), Naive Bayes (NB), extreme gradient boosting (XGBoost), and random forest (RF) machine learning models to classify the cancer types based on their tissue of origin. RF outperformed the other classifiers, with an average accuracy of 99%. Moreover, we applied local interpretable model-agnostic explanations (LIME) to explain important methylation biomarkers to classify cancer types.

Decidualized endometrial stromal cells present with altered androgen response in PCOS.

Hyperandrogenic women with PCOS show disrupted decidualization (DE) and placentation. Dihydrotestosterone (DHT) is reported to enhance DE in non-PCOS endometrial stromal cells (eSCCtrl); however, this has not been assessed in PCOS cells (eSCPCOS). Therefore, we studied the transcriptome profile of non-decidualized (non-DE) and DE eSCs from women with PCOS and Ctrl in response to short-term estradiol (E2) and/or progesterone (P4) exposure with/without (±) DHT. The non-DE eSCs were subjected to E2 ± DHT treatment, whereas the DE (0.5 mM 8-Br-cAMP, 96 h) eSCs were post-treated with E2 and P4 ± DHT, and RNA-sequenced. Validation was performed by immunofluorescence and immunohistochemistry. The results showed that, regardless of treatment, the PCOS and Ctrl samples clustered separately. The comparison of DE vs. non-DE eSCPCOS without DHT revealed PCOS-specific differentially expressed genes (DEGs) involved in mitochondrial function and progesterone signaling. When further adding DHT, we detected altered responses for lysophosphatidic acid (LPA), inflammation, and androgen signaling. Overall, the results highlight an underlying defect in decidualized eSCPCOS, present with or without DHT exposure, and possibly linked to the altered pregnancy outcomes. We also report novel factors which elucidate the mechanisms of endometrial dysfunction in PCOS.