Gut microbiome in endometriosis: a cohort study on 1000 individuals.

BACKGROUND: Endometriosis, defined as the presence of endometrial-like tissue outside of the uterus, is one of the most prevalent gynecological disorders. Although different theories have been proposed, its pathogenesis is not clear. Novel studies indicate that the gut microbiome may be involved in the etiology of endometriosis; nevertheless, the connection between microbes, their dysbiosis, and the development of endometriosis is understudied. This case-control study analyzed the gut microbiome in women with and without endometriosis to identify microbial targets involved in the disease. METHODS: A subsample of 1000 women from the Estonian Microbiome cohort, including 136 women with endometriosis and 864 control women, was analyzed. Microbial composition was determined by shotgun metagenomics and microbial functional pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Partitioning Around Medoids (PAM) algorithm was performed to cluster the microbial profile of the Estonian population. The alpha- and beta-diversity and differential abundance analyses were performed to assess the gut microbiome (species and KEGG orthologies (KO)) in both groups. Metagenomic reads were mapped to estrobolome-related enzymes' sequences to study potential microbiome-estrogen metabolism axis alterations in endometriosis. RESULTS: Diversity analyses did not detect significant differences between women with and without endometriosis (alpha-diversity: all p-values > 0.05; beta-diversity: PERMANOVA, both R 2 < 0.0007, p-values > 0.05). No differential species or pathways were detected after multiple testing adjustment (all FDR p-values > 0.05). Sensitivity analysis excluding women at menopause (> 50 years) confirmed our results. Estrobolome-associated enzymes' sequence reads were not significantly different between groups (all FDR p-values > 0.05). CONCLUSIONS: Our findings do not provide enough evidence to support the existence of a gut microbiome-dependent mechanism directly implicated in the pathogenesis of endometriosis. To the best of our knowledge, this is the largest metagenome study on endometriosis conducted to date.

The female upper reproductive tract harbors endogenous microbial profiles.

Introduction: The female reproductive tract harbours unique microbial communities (known as microbiota) which have been associated with reproductive functions in health and disease. While endometrial microbiome studies have shown that the uterus possesses higher bacterial diversity and richness compared to the vagina, the knowledge regarding the composition of the Fallopian tubes (FT) is lacking, especially in fertile women without any underlying conditions. Methods: To address this gap, our study included 19 patients who underwent abdominal hysterectomy for benign uterine pathology, and 5 women who underwent tubal ligation as a permanent contraceptive method at Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA). We analyzed the microbiome of samples collected from the FT and endometrium using 16S rRNA gene sequencing. Results: Our findings revealed distinct microbiome profiles in the endometrial and FT samples, indicating that the upper reproductive tract harbors an endogenous microbiome. However, these two sites also shared some similarities, with 69% of the detected taxa Being common to both. Interestingly, we identified seventeen bacterial taxa exclusively present in the FT samples, including the genera Enhydrobacter, Granulicatella, Haemophilus, Rhizobium, Alistipes, and Paracoccus, among others. On the other hand, 10 bacterial taxa were only found in the endometrium, including the genera Klebsiella, Olsenella, Oscillibacter and Veillonella (FDR <0.05). Furthermore, our study highlighted the influence of the endometrial collection method on the findings. Samples obtained transcervically showed a dominance of the genus Lactobacillus, which may indicate potential vaginal contamination. In contrast, uterine samples obtained through hysterescopy revealed higher abundance of the genera Acinetobacter, Arthrobacter, Coprococcus, Methylobacterium, Prevotella, Roseburia, Staphylococcus, and Streptococcus. Discussion: Although the upper reproductive tract appears to have a low microbial biomass, our results suggest that the endometrial and FT microbiome is unique to each individual. In fact, samples obtained from the same individual showed more microbial similarity between the endometrium and FT compared to samples from different women. Understanding the composition of the female upper reproductive microbiome provides valuable insights into the natural microenvironment where processes such as oocyte fertilization, embryo development and implantation occur. This knowledge can improve in vitro fertilization and embryo culture conditions for the treatment of infertility.

Endometrial whole metabolome profile at the receptive phase: influence of Mediterranean Diet and infertility.

Introduction: Several metabolite classes have been identified in human endometrium, including lipids, nucleotides, amino acids, organic acids, and sugars. The first studies suggest the importance of metabolites in endometrial functions, as imbalance in uterine metabolites has been associated with low implantation rate and endometriosis. Nevertheless, most of studies have put emphasis on specific metabolite classes, and we lack the knowledge of the whole metabolome composition in human uterus. Further, a healthy dietary pattern has been shown to potentially protect against different endometrial dysfunctions and is a potential modulator of metabolomic composition and, consequently, the intrauterine microenvironment. The Mediterranean Diet (MD), characterized by a high intake of fruits, vegetables, cereals, nuts, legumes, fish, and olive oil, and a low consumption of meat, dairy products, and processed foods, has been associated with a wide range of benefits for health. Indeed, the MD pattern has displayed a beneficial role in endometriosis management and fertility; however, the relationship between the MD and the endometrial metabolome is still unknown. In our study, we set out to analyze receptive-phase endometrial metabolome profiles among women with infertility and their associations with MD. Methods: The study included women with male factor infertility (n=8), unexplained infertility (n=10), recurrent implantation failure (n=14), and endometriosis (n=13). The endometrial metabolome was analyzed with ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). The MD adherence of the participants was assessed using the 14-point MEDAS questionnaire of adherence to the MD. Results: We provide the whole metabolome profile of the endometrium, where 925 different metabolites were identified. Among these metabolites, lipids comprised the largest part, where polyunsaturated fatty acids (PUFAs) prevailed. Women with endometriosis and recurrent implantation failure were found to have lower levels of PUFAs compared to women with male factor and unexplained infertility (i.e., no clear endometrial alterations), identifying a metabolome profile associated with infertility diagnoses where altered endometrial functions are suspected. Moreover, MD adherence seemed to be associated with the endometrial metabolomic profile in a manner dependent on the health status of the uterus. Conclusion: The study findings provide insight into the molecular background of female infertility and lead to identification of potential molecular biomarkers and possibilities for modulating the endometrial microenvironment and, thereby, endometrial functions involved in embryo implantation and infertility.

Microbial composition across body sites in polycystic ovary syndrome: a systematic review and meta-analysis.

Polycystic ovary syndrome (PCOS) is an endocrine disorder affecting reproductive-aged women, but the cause remains unclear. Recent evidence has linked microbial composition with PCOS; however, the results are inconsistent. The aim of this systematic review was to gather current knowledge of the microbes across body sites (oral cavity, blood, vagina/cervix, gut) in women with PCOS, and meta-analyse the microbial diversity in PCOS. For this purpose, a systematic search using PubMed, Web of Science, Cochrane and Scopus was carried out. After selection, 34 studies met the inclusion criteria. Most of the studies associated changes in the microbiome with PCOS, whereas heterogeneity of the studies in terms of ethnicity, body mass index (BMI) and methodology, among other confounders, made it difficult to corroborate this relationship. In fact, 19 out of 34 of the studies were categorised as having high risk of bias when the quality assessment was conducted. Our meta-analysis on the gut microbiome of 14 studies demonstrated that women with PCOS possess significantly lower microbial alpha diversity compared with controls (SMD = -0.204; 95% CI -0.360 to -0.048; P = 0.010; I2 = 5.508, by Shannon Index), which may contribute to the development of PCOS. Nevertheless, future studies should specifically overcome the shortcomings of the current studies by through well planned and conducted studies with larger sample sizes, proper negative and positive controls and adequate case-control matching.

The Upper Reproductive System Microbiome: Evidence beyond the Uterus.

The microbiome of the female upper reproductive system has garnered increasing recognition and has become an area of interest in the study of women's health. This intricate ecosystem encompasses a diverse consortium of microorganisms (i.e., microbiota) and their genomes (i.e., microbiome) residing in the female upper reproductive system, including the uterus, the fallopian tubes, and ovaries. In recent years, remarkable advancements have been witnessed in sequencing technologies and microbiome research, indicating the potential importance of the microbial composition within these anatomical sites and its impact in women's reproductive health and overall well-being. Understanding the composition, dynamics, and functions of the microbiome of the female upper reproductive system opens up exciting avenues for improving fertility, treating gynecological conditions, and advancing our comprehension of the intricate interplay between the microbiome and the female reproductive system. The aim of this study is to compile currently available information on the microbial composition of the female upper reproductive system in humans, with a focus beyond the uterus, which has received more attention in recent microbiome studies compared with the fallopian tubes and ovaries. In conclusion, this review underscores the potential role of this microbiome in women's physiology, both in health and disease.

Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets.

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males.

Physicochemical and Functional Characterization of Female Reproductive Fluids: A Report of the First Two Infants Born Following Addition of Their Mother's Fluids to the Embryo Culture Media.

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

Nitrite and Nitrate Levels in Follicular Fluid From Human Oocyte Donors Are Related to Ovarian Response and Embryo Quality.

Nitric oxide, a key regulatory molecule in the follicular fluid, has been suggested as a possible biomarker to predict ovarian response in stimulated cycles and the potential of the retrieved oocytes for developing high-quality embryos. Nevertheless, a consensus on whether or not nitric oxide can help in this context has not been reached. We simultaneously measured the oxidation products of nitric oxide, nitrite, and nitrate, via high-performance liquid chromatography (HPLC)-UV in follicular fluid samples from 72 oocyte donors. We found no associations of follicular fluid nitrite, nitrate, total nitric oxide, or nitrate/nitrite ratio with total or metaphase II (MII) oocyte yield. However, nitrite and nitrate levels were related to the yield of MII oocytes when this outcome was expressed as a proportion of all oocytes retrieved. The adjusted MII proportion in the lowest and highest nitrite levels were 68% (58-77%) and 79% (70-85%), respectively (p, linear trend = 0.02), whereas the adjusted MII proportion in extreme tertiles of nitrate levels were 79% (70-85%) and 68% (57-77%) (p, linear trend = 0.03). In addition, nitrate levels showed a suggestive inverse correlation with embryos with maximum or high potential of implantation (p = 0.07). These results suggest that the follicular fluid concentrations of nitrite and nitrate may be a useful tool in predicting how healthy oocyte donors respond to superovulation and the implantation potential of the embryos produced from their oocytes.

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies.

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation.

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads.

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte's shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.

Which Low-Abundance Proteins are Present in the Human Milieu of Gamete/Embryo Maternal Interaction?

The improvement of the embryo culture media is of high relevance due to its influence on successful implantation rates, pregnancy, neonatal outcomes, and potential effects in adult life. The ideal conditions for embryo development are those naturally occurring in the female reproductive tract, i.e., the oviductal and uterine fluids. To shed light on the differences between chemical and natural media, we performed the first comparative study of the low abundance proteins in plasma, uterine, and oviductal fluid collected, simultaneously, from healthy and fertile women that underwent a salpingectomy. The rationale for this design derives from the fact that high-abundant proteins in these fluids are usually those coming from blood serum and frequently mask the detection of low abundant proteins with a potentially significant role in specific processes related to the embryo-maternal interaction. The proteomic analysis by 1D-nano LC ESI-MSMS detected several proteins in higher amounts in oviductal fluid when compared to uterine and plasma samples (RL3, GSTA1, EZRI, DPYSL3, GARS, HSP90A). Such oviductal fluid proteins could be a target to improve fertilization rates and early embryo development if used in the culture media. In conclusion, this study presents a high-throughput analysis of female reproductive tract fluids and contributes to the knowledge of oviductal and uterine secretome.

Supplementation of bovine follicular fluid during in vitro maturation increases oocyte cumulus expansion, blastocyst developmental kinetics, and blastocyst cell number.

Bovine follicular fluid (bFF) is the natural milieu for oocyte growth and development. However, its value as supplementation to in vitro maturation medium is still questioned due to inconsistent results. In this study we hypothesized that adding 10% of follicular fluid as well as heat treating it to inhibit the complement system, would produce higher quality embryos. To do so, experiments were conducted to compare the effect of bFF and heat-treated bFF (bFFin) on oocyte competence assessed by different parameters such as nuclear and cytoplasmic maturation, IVF efficiency, in vitro embryo development and embryo survivability post-vitrification. No differences on nuclear maturation nor cortical granules migration were observed but differences were found on oocyte's cumulus cell expansion, with bFF group having the highest increase (79.0 ±â€¯3.7%). bFFin had a negative impact on IVF efficiency (58.6 ±â€¯3.2%), but no differences were found between bFF (62.9 ±â€¯3.2%) and control (72.8 ±â€¯3.0%). Although the cleavage and blastocyst rate were similar between groups, the day 6 embryo development rate was higher in bFFin group, suggesting an accelerated developmental kinetics. Hatched blastocysts from the bFF group showed a higher cell count than the control group (241.3 ±â€¯20.1 and 185.8 ±â€¯10.0, respectively), and bFFin embryos showed values in between (214.9 ±â€¯14.0). No difference on survivability post-vitrification was found between groups, although the blastocyst stage had a significant impact on the survival rate across all groups. In conclusion, using bFF as supplementation to maturation medium showed a higher benefit when comparing to the standard supplementation by having oocytes with higher cumulus expansion rate, faster development of embryos and higher number of cells per embryo. Inactivation of bFF lowered IVF efficiency but didn't compromise blastocyst development and quality.

Hierarchical scaffolds enhance osteogenic differentiation of human Wharton's jelly derived stem cells.

Hierarchical structures, constituted by polymeric nano and microfibers, have been considered promising scaffolds for tissue engineering strategies, mainly because they mimic, in some way, the complexity and nanoscale detail observed in real organs. The chondrogenic potential of these scaffolds has been previously demonstrated, but their osteogenic potential is not yet corroborated. In order to assess if a hierarchical structure, with nanoscale details incorporated, is an improved scaffold for bone tissue regeneration, we evaluate cell adhesion, proliferation, and osteogenic differentiation of human Wharton's jelly derived stem cells (hWJSCs), seeded into hierarchical fibrous scaffolds. Biological data corroborates that hierarchical fibrous scaffolds show an enhanced cell entrapment when compared to rapid prototyped scaffolds without nanofibers. Furthermore, upregulation of bone specific genes and calcium phosphate deposition confirms the successful osteogenic differentiation of hWJSCs on these scaffolds. These results support our hypothesis that a scaffold with hierarchical structure, in conjugation with hWJSCs, represents a possible feasible strategy for bone tissue engineering applications.