Gut bacteriome and mood disorders in women with PCOS.

STUDY QUESTION: How does the gut bacteriome differ based on mood disorders (MDs) in women with polycystic ovary syndrome (PCOS), and how can the gut bacteriome contribute to the associations between these two conditions? SUMMARY ANSWER: Women with PCOS who also have MDs exhibited a distinct gut bacteriome with reduced alpha diversity and a significantly lower abundance of Butyricicoccus compared to women with PCOS but without MDs. WHAT IS KNOWN ALREADY: Women with PCOS have a 4- to 5-fold higher risk of having MDs compared to women without PCOS. The gut bacteriome has been suggested to influence the pathophysiology of both PCOS and MDs. STUDY DESIGN, SIZE, DURATION: This population-based cohort study was derived from the Northern Finland Birth Cohort 1966 (NFBC1966), which includes all women born in Northern Finland in 1966. Women with PCOS who donated a stool sample at age 46 years (n = 102) and two BMI-matched controls for each case (n = 205), who also responded properly to the MD criteria scales, were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 102 women with PCOS and 205 age- and BMI-matched women without PCOS were included. Based on the validated MD criteria, the subjects were categorized into MD or no-MD groups, resulting in the following subgroups: PCOS no-MD (n = 84), PCOS MD (n = 18), control no-MD (n = 180), and control MD (n = 25). Clinical characteristics were assessed at age 31 years and age 46 years, and stool samples were collected from the women at age 46 years, followed by the gut bacteriome analysis using 16 s rRNA sequencing. Alpha diversity was assessed using observed features and Shannon's index, with a focus on genera, and beta diversity was characterized using principal components analysis (PCA) with Bray-Curtis Dissimilarity at the genus level. Associations between the gut bacteriome and PCOS-related clinical features were explored by Spearman's correlation coefficient. A P-value for multiple testing was adjusted with the Benjamini-Hochberg false discovery rate (FDR) method. MAIN RESULTS AND THE ROLE OF CHANCE: We observed changes in the gut bacteriome associated with MDs, irrespective of whether the women also had PCOS. Similarly, PCOS MD cases showed a lower alpha diversity (Observed feature, PCOS no-MD, median 272; PCOS MD, median 208, FDR = 0.01; Shannon, PCOS no-MD, median 5.95; PCOS MD, median 5.57, FDR = 0.01) but also a lower abundance of Butyricicoccus (log-fold changeAnalysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC)=-0.90, FDRANCOM-BC=0.04) compared to PCOS no-MD cases. In contrast, in the controls, the gut bacteriome did not differ based on MDs. Furthermore, in the PCOS group, Sutterella showed positive correlations with PCOS-related clinical parameters linked to obesity (BMI, r2=0.31, FDR = 0.01; waist circumference, r2=0.29, FDR = 0.02), glucose metabolism (fasting glucose, r2=0.46, FDR < 0.001; fasting insulin, r2=0.24, FDR = 0.05), and gut barrier integrity (zonulin, r2=0.25, FDR = 0.03). LIMITATIONS, REASONS FOR CAUTION: Although this was the first study to assess the link between the gut bacteriome and MDs in PCOS and included the largest PCOS dataset for the gut microbiome analysis, the number of subjects stratified by the presence of MDs was limited when contrasted with previous studies that focused on MDs in a non-selected population. WIDER IMPLICATIONS OF THE FINDINGS: The main finding is that gut bacteriome is associated with MDs irrespective of the PCOS status, but PCOS may also modulate further the connection between the gut bacteriome and MDs. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie Grant Agreement (MATER, No. 813707), the Academy of Finland (project grants 315921, 321763, 336449), the Sigrid Jusélius Foundation, Novo Nordisk Foundation (NNF21OC0070372), grant numbers PID2021-12728OB-100 (Endo-Map) and CNS2022-135999 (ROSY) funded by MCIN/AEI/10.13039/501100011033 and ERFD A Way of Making Europe. The study was also supported by EU QLG1-CT-2000-01643 (EUROBLCS) (E51560), NorFA (731, 20056, 30167), USA/NIH 2000 G DF682 (50945), the Estonian Research Council (PRG1076, PRG1414), EMBO Installation (3573), and Horizon 2020 Innovation Grant (ERIN, No. EU952516). The funders did not participate in any process of the study. We have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Chromatin modifier developmental pluripotency associated factor 4 (DPPA4) is a candidate gene for alcohol-induced developmental disorders.

BACKGROUND: Prenatal alcohol exposure (PAE) affects embryonic development, causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neuronal disorders and birth defects. We hypothesize that early alcohol-induced epigenetic changes disrupt the accurate developmental programming of embryo and consequently cause the complex phenotype of developmental disorders. To explore the etiology of FASD, we collected unique biological samples of 80 severely alcohol-exposed and 100 control newborns at birth. METHODS: We performed genome-wide DNA methylation (DNAm) and gene expression analyses of placentas by using microarrays (EPIC, Illumina) and mRNA sequencing, respectively. To test the manifestation of observed PAE-associated DNAm changes in embryonic tissues as well as potential biomarkers for PAE, we examined if the changes can be detected also in white blood cells or buccal epithelial cells of the same newborns by EpiTYPER. To explore the early effects of alcohol on extraembryonic placental tissue, we selected 27 newborns whose mothers had consumed alcohol up to gestational week 7 at maximum to the separate analyses. Furthermore, to explore the effects of early alcohol exposure on embryonic cells, human embryonic stem cells (hESCs) as well as hESCs during differentiation into endodermal, mesodermal, and ectodermal cells were exposed to alcohol in vitro. RESULTS: DPPA4, FOXP2, and TACR3 with significantly decreased DNAm were discovered-particularly the regulatory region of DPPA4 in the early alcohol-exposed placentas. When hESCs were exposed to alcohol in vitro, significantly altered regulation of DPPA2, a closely linked heterodimer of DPPA4, was observed. While the regulatory region of DPPA4 was unmethylated in both control and alcohol-exposed hESCs, alcohol-induced decreased DNAm similar to placenta was seen in in vitro differentiated mesodermal and ectodermal cells. Furthermore, common genes with alcohol-associated DNAm changes in placenta and hESCs were linked exclusively to the neurodevelopmental pathways in the enrichment analysis, which emphasizes the value of placental tissue when analyzing the effects of prenatal environment on human development. CONCLUSIONS: Our study shows the effects of early alcohol exposure on human embryonic and extraembryonic cells, introduces candidate genes for alcohol-induced developmental disorders, and reveals potential biomarkers for prenatal alcohol exposure.

Endometriosis and irritable bowel syndrome: similarities and differences in the spectrum of comorbidities.

STUDY QUESTION: Do the spectrum and prevalence of comorbidities of endometriosis and irritable bowel syndrome (IBS) overlap? SUMMARY ANSWER: Despite several overlapping symptoms, the most significantly associated comorbidities of endometriosis and IBS are different and are rather related to the organ systems primarily involved in the index diagnosis. WHAT IS KNOWN ALREADY: Endometriosis and IBS both have several similar unspecific symptoms, such as recurrent abdominal pain, cramping and anxiety, and both diseases affect young women and are associated with a number of comorbidities causing a poor quality of life. However, a detailed study, revealing the full spectrum of endometriosis and IBS comorbidities in the same study population, is lacking. STUDY DESIGN, SIZE, DURATION: This article presents a retrospective in silico analysis of the data from a large nationwide biobank-based cohort consisting of 121 773 women. After excluding all first- and second-degree relatives, the data of up to 65 421 women were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: International Classification of Disease-10 diagnosis main codes associated with endometriosis (N80) and IBS (K58) diagnoses were identified from the Estonian Biobank dataset by linking with the Estonian Health Insurance Fund and other relevant registries. The associations between N80 and K58 and other diagnosis codes were tested using logistic regression, adjusting for age at recruitment and 10 genetic principal components to account for potential population stratification. Bonferroni correction was applied to account for multiple testing. MAIN RESULTS AND THE ROLE OF CHANCE: Both women with endometriosis and IBS suffered from more conditions compared to the control group, with 226 and 428 diagnosis codes statistically significantly more frequent in women with respective diagnosis compared to controls. Women suffering from both conditions had 275 significantly associated comorbidities. A remarkable proportion of women with IBS or endometriosis suffered also from endometriosis (9.0%) or IBS (13.6%), respectively. In endometriosis, the most prevalent diagnoses were related to diseases of the genitourinary system (33 N-category codes) and in women with IBS, the most associated diagnoses were related to digestive disorders and gastrointestinal tract (52 codes from K-category). Among the most significant diagnoses in endometriosis were uterine leiomyomas (D25), menstrual disorders (N92) and infertility (N97) (P < 1 × 10-315 for all), and in IBS, lactose intolerance (E73), gastritis and duodenitis (K29) and functional dyspepsia (K30) were in the top list of most significant comorbidities (P < 1 × 10-315 for all). LIMITATIONS, REASONS FOR CAUTION: The information about the severity stages of endometriosis and subtypes of IBS was not available for analysis. The findings may not be fully extrapolated to all female populations, because all participants were from one geographic area and had good access to health services. WIDER IMPLICATIONS OF THE FINDINGS: These findings support previous studies that have found a high prevalence of pre-selected comorbidities in women with endometriosis and IBS. However, taking into account the differences in the full spectrum of comorbidities of endometriosis and IBS may aid in diagnosing these disorders. Women and healthcare providers need to be aware that women with endometriosis are at high risks of complications during pregnancy and should be carefully monitored. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Estonian Research Council (grant PRG1076), Horizon 2020 innovation grant (ERIN, grant no. EU952516), Enterprise Estonia (grant no. EU48695), MSCA-RISE-2020 project TRENDO (grant no. 101008193) and by the European Union through the European Regional Development Fund (Projects no. 2014-2020.4.01.15-0012 and no. 2014-2020.4.01.16-0125). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Copy number variation analysis detects novel candidate genes involved in follicular growth and oocyte maturation in a cohort of premature ovarian failure cases.

STUDY QUESTION: Can spontaneous premature ovarian failure (POF) patients derived from population-based biobanks reveal the association between copy number variations (CNVs) and POF? SUMMARY ANSWER: CNVs can hamper the functional capacity of ovaries by disrupting key genes and pathways essential for proper ovarian function. WHAT IS KNOWN ALREADY: POF is defined as the cessation of ovarian function before the age of 40 years. POF is a major reason for female infertility, although its cause remains largely unknown. STUDY DESIGN, SIZE, DURATION: The current retrospective CNV study included 301 spontaneous POF patients and 3188 control individuals registered between 2003 and 2014 at Estonian Genome Center at the University of Tartu (EGCUT) biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA samples from 301 spontaneous POF patients were genotyped by Illumina HumanCoreExome (258 samples) and HumanOmniExpress (43 samples) BeadChip arrays. Genotype and phenotype information was drawn from the EGCUT for the 3188 control population samples, previously genotyped with HumanCNV370 and HumanOmniExpress BeadChip arrays. All identified CNVs were subjected to functional enrichment studies for highlighting the POF pathogenesis. Real-time quantitative PCR was used to validate a subset of CNVs. Whole-exome sequencing was performed on six patients carrying hemizygous deletions that encompass genes essential for meiosis or folliculogenesis. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven novel microdeletions and microduplications that encompass genes relevant to POF were identified. For example, FMN2 (1q43) and SGOL2 (2q33.1) are essential for meiotic progression, while TBP (6q27), SCARB1 (12q24.31), BNC1 (15q25) and ARFGAP3 (22q13.2) are involved in follicular growth and oocyte maturation. The importance of recently discovered hemizygous microdeletions of meiotic genes SYCE1 (10q26.3) and CPEB1 (15q25.2) in POF patients was also corroborated. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis and no functional studies were performed. Anamnestic data obtained from population-based biobank lacked clinical, biological (hormone levels) or ultrasonographical data, and spontaneous POF was predicted retrospectively by excluding known extraovarian causes for premature menopause. WIDER IMPLICATIONS OF THE FINDINGS: The present study, with high number of spontaneous POF cases, provides novel data on associations between the genomic aberrations and premature menopause of ovarian cause and demonstrates that population-based biobanks are powerful source of biological samples and clinical data to reveal novel genetic lesions associated with human reproductive health and disease, including POF. STUDY FUNDING/COMPETING INTEREST: This study was supported by the Estonian Ministry of Education and Research (IUT20-43, IUT20-60, IUT34-16, SF0180027s10 and 9205), Enterprise Estonia (EU30020 and EU48695), Eureka's EUROSTARS programme (NOTED, EU41564), grants from European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, |EU324509) and Horizon 2020 innovation programme (WIDENLIFE, 692065), Academy of Finland and the Sigrid Juselius Foundation.

Single-cell transcriptome analysis of endometrial tissue.

STUDY QUESTION: How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER: By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY: Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION: The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION: Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS: The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level.

The prevalence and phenotypic characteristics of spontaneous premature ovarian failure: a general population registry-based study.

STUDY QUESTION: What is the measured prevalence and phenotype of spontaneous premature ovarian failure (POF) in the general population? SUMMARY ANSWER: Spontaneous POF occurs in ∼1% of the general population with unique phenotype of post-menopausal ageing distinct from surgically induced premature menopause. WHAT IS KNOWN ALREADY: POF is multifactorial ovarian quiescence before the age of 40. The clinical features of POF are diverse and the population prevalence of POF is still not known. STUDY DESIGN, SIZE, DURATION: This population-depictive registry-based case-cohort study included 34 041 women from the Estonian Genome Center registered between 2003 and 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spontaneous POF was selected retrospectively by excluding other causes for premature menopause under the age of 40 (N = 310) and women with surgically induced premature menopause participated as a reference group (N = 242). MAIN RESULTS AND THE ROLE OF CHANCE: The prevalence of spontaneous POF was 0.91% (0.81-1.02%) among women of the general population in Estonia. In women with POF, menarche occurred a few months later than in the reference group and a significantly higher number of live births during their reproductive life was recorded. Women with POF also consumed less alcohol and had smaller waist-to-hip ratios than those in the reference group, although both groups of women were similar in body mass index a decade after menopause. The prevalence of concomitant diseases was similar between two groups of women by their fifties, but the pattern of onset of these diseases was different. Surgically induced premature menopause associated with faster development of osteoporosis, hypertension, and connective tissue diseases, but slower development of allergies, compared with spontaneous POF. The age of menopause was determined by irregular menstrual cycles, but not by the length of regular menstrual cycles, the age of menarche, the number of pregnancies or live births, smoking or alcohol consumption, or the use of oral contraceptives for some time during the reproductive period. LIMITATIONS, REASONS FOR CAUTION: POF is rarely stated in medical records and cannot be diagnosed retrospectively by standard procedures. Therefore the data on all cases of women with primary amenorrhea or premature menopause before the age of 40 were requested from the registry and spontaneous POF was predicted retrospectively by excluding other extraovarian causes for premature menopause. Since the current study is retrospective registry-based data analysis, no genetic evaluation concerning possible candidate genes and no blood analysis concerning immunologic disorders could be performed to describe etiopathogenesis of POF. WIDER IMPLICATION OF THE FINDINGS: Spontaneous POF most likely comprises several diseases with different etiopathologies and there may be a unique phenotype of post-menopausal ageing distinct from that in surgically induced premature menopause. Irregular menstrual cycles may be a prospective risk for developing spontaneous POF. Compared with spontaneous POF, surgically induced premature menopause associates with faster development of age-related diseases. The data point to new ideas and hypotheses for further studies on etiopathologies and treatment options for spontaneous POF. STUDY FUNDING/COMPETING INTERESTS: The study was funded by grant SF0180044s09, SF0180027s10 and IUT20-43 from the Estonian Ministry of Education and Research, Enterprise Estonia, grant no EU30020, Eureka's EUROSTARS programme grant (NOTED, EU41564). No competing interests are declared.

Systemic oxidative stress could predict assisted reproductive technique outcome.

PURPOSE: Previous studies have indicated that OxS (oxidative stress) may appear as a possible reason for poor ART outcome. Our aim was to study OxS levels in both partners of couples seeking Assisted reproduction Technology (ART). METHODS: Altogether 79 couples were recruited. Oxidative DNA damage (8-OHdG) and lipid peroxidation (8-EPI) were measured, and clinical background and ART outcomes were recorded. RESULTS: Both OxS markers accurately reflected clinical conditions with prominent negative effects attributable to genital tract infections, endometriosis, uterine myoma and smoking. Furthermore, the level of OxS was also affected by partner's state of health. The highest 8-EPI levels were detected in both partners when biochemically detectable pregnancies did not develop into clinically detectable pregnancies (in women, 97,8 ± 16,7 vs 72.9 ± 22,9, p = 0.007; in men, 89.6 ± 20,4 vs 72,1 ± 22,6, p = 0.049). CONCLUSIONS: To conclude, high grade systemix OxS in both partners may negatively affect the maintenance and outcome of pregnancy. Applying the detection of OxS in ART patients may select patients with higher success rate and/or those who require antioxidant therapy. This would lead to improvement of ART outcome as well as natural fertility.

Folic acid supplementation and IVF pregnancy outcome in women with unexplained infertility.

Folic acid supplements are commonly used by infertile women which leads to a positive folate status. However, the effect of folic acid supplements on pregnancy outcome in women with unexplained infertility has not been well investigated. This study evaluated folic acid supplement use and folate status in women with unexplained infertility in relation to IVF pregnancy outcome. In addition, use of folic acid supplements and folate status were compared between women with unexplained infertility and fertile, nonpregnant control women. Women with unexplained infertility used significantly more folic acid supplements and had higher median total folic acid intake from supplements compared with fertile control women (both P < 0.001). Women with unexplained infertility also had significantly higher median plasma folate and lower median plasma homocysteine concentrations than fertile women (both P < 0.001), but folic acid supplementation or folate status were not related to pregnancy outcome in women with unexplained infertility. In conclusion, folic acid supplementation or good folate status did not have a positive effect on pregnancy outcome following infertility treatment in women with unexplained infertility. Folate is one of the B vitamins which has been suggested to be related to infertility. Folic acid is an artificial form of folate which is commonly used in dietary supplements. Folic acid supplementation has been shown to increase folate concentrations and decrease concentrations of the amino acid homocysteine in the blood. Folic acid supplementation is commonly used by infertile women, but the effect on pregnancy outcome in women with a diagnosis of unexplained infertility has not been thoroughly investigated. In the present study, folic acid supplement use and folate status (concentrations of folate and homocysteine) in women with unexplained infertility were evaluated in relation to pregnancy outcome. In addition, the use of folic acid supplements and folate status were compared between women with unexplained infertility and fertile control women. Our results showed that women with unexplained infertility used considerably more folic acid supplements and had higher total folic acid intake from supplements compared with fertile control women. Women with unexplained infertility had better blood folate and homocysteine concentrations than fertile women, but folic acid supplementation or folate status were not related to pregnancy outcome following the infertility treatment. In conclusion, high folic acid intake or good folate status did not increase the possibility of a birth of a healthy baby after infertility treatment in women with unexplained infertility.

Impact of Lactobacillus crispatus-containing oral and vaginal probiotics on vaginal health: a randomised double-blind placebo controlled clinical trial.

Health of reproductive tract is tightly associated with balance of microbial communities in this area. Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) represent common disturbances of vaginal communities. Vaginal discharge due to BV or VVC is a very frequent reason for visiting gynaecologist. We aimed to evaluate the impact of the novel evidence-based probiotics on BV and VVC patients. The study group included 89 BV and 93 VVC patients (aged 18-50 years) who were recruited into randomised double-blind placebo-controlled two-arm parallel trial. The patients of each diagnosis group received oral or vaginal probiotic capsules, or placebo capsules during 3 months. A probiotic capsule contained two (DSM32717 and DSM32720, in case of BV) or three (DSM32720, DSM32718 and DSM32716, in case of VVC) Lactobacillus crispatus strains. Vaginal, intestinal and general health was monitored weekly by questionnaire. Blood analyses were done in the beginning and at the end of trial. Vaginal samples were collected monthly, microscopic and molecular analyses were performed. The study revealed that both oral and vaginal capsules reduced the signs and symptoms in BV patients. Remarkable improvement was noted in Nugent score, amount and smell of discharge, but also in itching/irritation. Consumption of vaginal probiotics significantly increased the lactobacilli counts in their vagina while mean proportion of some BV-related bacteria decreased. In VVC patients, both oral and vaginal capsules lowered the combined score of two most important symptoms, amount of discharge and itching/irritation. In conclusion, the novel formulations of evidence-based well-focused probiotic L. crispatus strains are effective against BV and VVC being suitable for both vaginal and oral administration. Clinical trial registration: ISRCTN34840624, BioMed Central.

No evidence of somatic DNA copy number alterations in eutopic and ectopic endometrial tissue in endometriosis.

BACKGROUND: De novo somatic copy number aberrations (SCNAs) in eutopic and ectopic endometria are thought to be involved in the pathogenesis of endometriosis. In this study we used, for the first time, high-density single nucleotide polymorphism-array technology for accurate detection of SCNAs, inherited DNA copy number variations (CNVs) and copy-neutral loss of heterozygosity (cn-LOH) patterns in patients with endometriosis. METHODS: The Illumina HumanOmniExpress array was used to detect de novo somatic genomic alterations in eutopic and ectopic endometria from 11 women (eight with Stage I-II endometriosis and three with Stage III-IV endometriosis) by comparatively analysing DNA from peripheral blood, eutopic endometrium and a pure population of endometriotic cells harvested from endometriotic lesions by laser capture microdissection (LCM). The frequency of the CNV in 3p14.1 from blood DNA of 187 endometriosis patients (94 with Stage I-II endometriosis and 93 with Stage III-IV endometriosis) and 171 healthy women from the Estonian general population was evaluated. RESULTS: Analysis of array data showed that LCM DNA can be used successfully for detection of genetic changes as all inherited CNVs were identified in all tissues studied. No unique SCNAs or cases of cn-LOH were found in either eutopic or ectopic endometrium when compared with blood DNA. The frequency of the deletion allele in 3p14.1 did not differ between studied groups. CONCLUSIONS: In the present study no endometriosis-specific SCNAs or regions of cn-LOH in eutopic or ectopic endometrium were found. Nevertheless, as we studied only 17 endometriotic tissues derived from 11 patients we cannot entirely exclude the occurrence of rare SCNAs. Based on our results we suggest that molecular mechanisms other than chromosomal rearrangements most likely underlie the onset and progression of endometriosis.

The mid-secretory endometrial transcriptomic landscape in endometriosis: a meta-analysis.

STUDY QUESTION: Do women with endometriosis have a different endometrial gene expression profile at the time of embryo implantation than women without endometriosis? SUMMARY ANSWER: The endometrial gene expression profile of women with endometriosis differs from that of women without endometriosis at the mid-secretory phase, although the differences are small. WHAT IS KNOWN ALREADY: About 50% of women with endometriosis suffer infertility. Several molecular studies have suggested impaired endometrial receptivity in women with endometriosis, while others have detected no dysregulation of endometrial receptivity. Nevertheless, the previous endometrial transcriptome studies comparing women with and without endometriosis have been performed in small sample size with limited statistical power. We set out to systematically search and compile data of endometrial gene expression signatures at the receptive phase in women with endometriosis versus control women. Based on the obtained data, we conducted a meta-analysis of differentially expressed genes in order to raise the power of the analysis for identifying the molecular profiles of receptive phase endometria in endometriosis. STUDY DESIGN SIZE DURATION: A systematic literature search was conducted up to February 2022 following PRISMA criteria and included PubMed, Cochrane and Web of Science databases. For the systematic search, the term 'endometriosis' was paired with the terms 'transcriptomics', 'transcriptome', 'gene expression', 'RNA-seq', 'sequencing' and 'array', by using the Boolean operator 'AND' to connect them. Articles written in English were screened and interrogated for data extraction. PARTICIPANTS/MATERIALS SETTING METHODS: A meta-analysis was performed on the selected studies to extract the differentially expressed genes described at the mid-secretory phase in women with endometriosis versus women without endometriosis in natural cycles, using the robust rank aggregation method. In total, transcriptome data of 125 women (78 patients and 47 controls) were meta-analysed, with a special focus on endometrial receptivity-specific genes based on commercial endometrial receptivity tests. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 8 studies were eligible for the quantitative meta-analysis, gathering transcriptome data from the mid-secretory phase endometria of 125 women. A total of 7779 differentially expressed transcripts between the study groups were retrieved (3496 up-regulated and 4283 down-regulated) and were meta-analysed. After stringent multiple correction, there was no differential expression of any single molecule in the endometrium of women with endometriosis versus controls, while enrichment analysis detected that the pathways of chemotaxis and locomotion are dysregulated in endometriosis. Further analysis of endometrial receptivity-specific genes highlighted dysregulation of C4BPA, MAOA and PAEP and enrichment of immune and defence pathways in women with endometriosis. LIMITATIONS REASONS FOR CAUTION: Most of the studies included into the meta-analysis were relatively small and had different study designs, which might have contributed to a bias. WIDER IMPLICATIONS OF THE FINDINGS: The current meta-analysis supports the hypothesis that endometrial receptivity is altered in women with endometriosis, although the changes are small. The molecules and pathways identified could serve as future biomarkers and therapeutical targets in detecting and treating endometriosis-associated infertility. STUDY FUNDING/COMPETING INTERESTS: The authors declare no competing interests. This work was supported by the Spanish Ministry of Education, Culture and Sport [grant FPU15/01193] and the Margarita Salas program for the Requalification of the Spanish University system [grant UJAR01MS]; Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526-R; Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20); the Junta de Andalucía [BIO-302; and PAIDI P20_00158]; the University of Jaén [PAIUJA-EI_CTS02_2017]; the University of Granada, Plan Propio de Investigación 2016, Excellence actions: Units of Excellence; Unit of Excellence on Exercise and Health (UCEES), and by the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades and European Regional Development Fund (ERDF), ref. SOMM17/6107/UGR; the Estonian Research Council (grant PRG1076); Horizon 2020 innovation (ERIN, grant no. EU952516) of the European Commission and Enterprise Estonia (grant EU48695). TRIAL REGISTRATION NUMBER: The systematic review was registered at PROSPERO (identifier: CRD42020122054).

Folate-metabolizing gene variants and pregnancy outcome of IVF.

There is growing evidence that folate status and variation in folate-metabolizing genes are involved in female reproductive functions. This study evaluated the influence of maternal blood folate, vitamin B(12), homocysteine and 10 folate pathway gene variants on IVF outcome. Also, the prevalence of these polymorphisms was compared in 439 female IVF patients and 225 fertile controls. MTHFR 677 CT heterozygotes had a higher proportion of good-quality embryos and an increased chance of pregnancy. MTHFR 1793 GA heterozygosity was associated with a lower percentage of previously failed IVF treatments. Heterozygosity for FOLR1 1816 C/delC and 1841 G/A was associated with a raised risk of pregnancy loss. The CTH 1208 GT genotype was associated with an increased chance of pregnancy and a smaller number of previously failed IVF cycles and the genotype frequency was lower in IVF patients with three or more previously failed IVF treatments compared with fertile controls. SLC19A1 80 GA heterozygotes had a decreased number of previously failed IVF treatments and were more prevalent among fertile controls. In conclusion, polymorphisms in folate-metabolizing genes may affect ovarian stimulation and pregnancy outcome of IVF, and heterozygous individuals, rather than the wild-type homozygotes, appeared to have more favourable outcomes.

Growth characterization of individual rye sourdough bacteria by isothermal microcalorimetry.

AIMS: The present work tests the feasibility of the isothermal microcalorimetry method to study the performance of individual lactic acid bacteria during solid-state fermentation in rye sourdough. Another aim was to elucidate the key factors leading to the formation of different microbial consortia in laboratory and industrial sourdough during continuous backslopping propagation. METHODS AND RESULTS: Strains of the individual LAB isolated from industrial and laboratory sourdough cycle were grown in 10 kGy irradiated rye dough in vials of an isothermal calorimeter and the power-time curves were obtained. Sugars, organic acids and free amino acids in the sourdough were measured. The OD-time curves of the LAB strains during growth in flour extract or MRS (De Man, Rogosa and Sharpe) broth were also determined. The maximum specific growth rates of Lactobacillus sakei, Lactobacillus brevis, Lactobacillus curvatus and Leuconostoc citreum strains that dominated in backslopped laboratory sourdough were higher than those of Lactobacillus helveticus, Lactobacillus panis, Lactobacillus vaginalis, Lactobacillus casei and Lactobacillus pontis strains originating from industrial sourdough. Industrial strains had higher specific growth rates below pH 4·8. It was supposed that during long-run industrial backslopping processes, the oxygen sensitive species start to dominate because of the O(2) protective effect of rye sourdough. CONCLUSIONS: Measurements of the power-time curves revealed that the LAB strains dominating in the industrial sourdough cycle had better acid tolerance but lower maximum growth rate and oxygen tolerance than species isolated from a laboratory sourdough cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: Isothermal microcalorimetry combined with chemical analysis is a powerful method for characterization of sourdough fermentation process and determination of growth characteristics of individual bacteria in sourdough.

Endometrial gene expression analysis at the time of embryo implantation in women with unexplained infertility.

Successful embryo implantation depends on the quality of the embryo, as well as on the receptivity of the endometrium. The aim of this study was to investigate the endometrial gene expression profile in women with unexplained infertility in comparison with fertile controls at the time of embryo implantation in order to find potential predictive markers of uterine receptivity and to identify the molecular mechanisms of infertility. High-density oligonucleotide gene arrays, comprising 44 000 gene targets, were used to define the endometrial gene expression profile in infertile (n = 4) and fertile (n = 5) women during the mid-secretory phase (day LH + 7). Microarray results were validated using real-time PCR. Analyses of expression data were carried out using non-parametric methods. Hierarchical clustering and principal component analysis showed a clear distinction in endometrial gene expression between infertile and fertile women. In total we identified 145 significantly (>3-fold change) up-regulated and 115 down-regulated genes in infertile women versus controls. Via Database for Annotation, Visualization and Integrated Discovery functional analysis we detected a substantial number of dysregulated genes in the endometria of infertile women, involved in cellular localization (21.1%) and transport (18.8%) and transporter activity (13.1%) and with major localization in extracellular regions (19.2%). Ingenuity Pathways Analysis of the gene list showed dysregulation of gene pathways involved in leukocyte extravasation signalling, lipid metabolism and detoxification in the endometria of infertile women. In conclusion, endometrial gene expression in women with unexplained infertility at the time of embryo implantation is markedly different from that in fertile women. These results provide new information on genes and pathways that may have functional significance as regards to endometrial receptivity and subsequent embryo implantation.

The differential transcriptome and ontology profiles of floating and cumulus granulosa cells in stimulated human antral follicles.

Communication between various ovarian cell types is a prerequisite for folliculogenesis and ovulation. In antral follicles granulosa cells divide into two distinct populations of mural and cumulus granulosa cells (CGC), enveloping the antrum and surrounding the oocyte, respectively. Both cell types, with the mural compartment in excess, contribute to the floating granulosa cell (FGC) population in the follicular fluid. The aim of this study was to compare the transcriptomes of FGC and CGC in stimulated antral follicles obtained from 19 women undergoing IVF-ICSI procedure. FGC were obtained from follicular fluid during the follicle puncture procedure and CGC were acquired after oocyte denudation for micromanipulation. Gene expression analysis was conducted using the genome-wide Affymetrix transcriptome array. The expression profile of the two granulosa cell populations varied significantly. Out of 28 869 analysed transcripts 4480 were differentially expressed (q-value < 10(-4)) and 489 showed > or =2-fold difference in the expression level with 222 genes up-regulated in FGC and 267 in CGC. The transcriptome of FGC showed higher expression of genes involved in immune response, hematological system function and organismal injury, although CGC had genes involved in protein degradation and nervous system function up-regulated. Cell-to-cell signalling and interaction pathways were noted in both cell populations. Furthermore, numerous novel transcripts that have not been previously described in follicular physiology were identified. In conclusion, our results provide a solid basis for future studies in follicular biology that will help to identify molecular markers for oocyte and embryo viability in IVF.

Potential vaginal probiotics: safety, tolerability and preliminary effectiveness.

Vaginal discharge is one of the common reasons for gynaecologist consultation, as bacterial vaginosis and candidiasis are the main causes of discharge. These patients frequently experience numerous problems due to recurrent infections, side effects and drug resistance therefore alternative drugs are needed. Our primary aim was to evaluate safety and tolerability of the potentially probiotic Lactobacillus crispatus strains in volunteer women considering themselves healthy. We also monitored the effects of these strains on vaginal health parameters and lactobacilli counts in vagina and intestine. Forty women were recruited into trial. Absence of chronic diseases was confirmed by questionnaire and blood analysis at screening visit. In randomised double-blind placebo-controlled crossover study the eligible participants were randomly allocated to one of four groups and had to consume one of the two study products (Pro I or Pro II) - a capsule containing 3 strains, 109 cfu per strain, or placebo for 1 week. Treatment period was followed by 2-week washout period and continued with second treatment and washout period. Individuals receiving firstly probiotic, received later placebo and vice versa. Blood, vaginal and faecal samples were collected, and self-reported questionnaires were applied. Thirty subjects completed the trial. The probiotic capsules were well-tolerated. The Pro II intake resulted in a significant decrease in Nugent score (from median 3.0 to 2.0, mean 3.9 to 2.6, P=0.002) and reduction in Gardnerella vaginalis counts (log10 3.57 to 2.38; P=0.027). Reduction of total vaginal bacterial counts was revealed in Pro I group (log10 7.99 to 7.72; P=0.048). In conclusion, the selected vaginal L. crispatus strains are well tolerable and Pro II mixture is prospectively effective in reducing Nugent score and vaginal counts of G. vaginalis. Therefore, these strains seem to be promising candidates for development of novel evidence-based well-focused probiotics to target female urogenital tract disorders.

Fine-scale quantification of HCG beta gene transcription in human trophoblastic and non-malignant non-trophoblastic tissues.

Human chorionic gonadotropin (HCG) is produced by syncytiotrophoblast of placenta. It delays the apoptosis of corpus luteum and functions in implantation. Its possible role in male reproduction has been raised. HCG beta subunit is encoded by CGB, CGB5, CGB7 and CGB8 genes located at 19q13.3 in a common genome cluster with beta subunit non-coding CGB1 and CGB2. We conducted a sensitive quantification and comparison of CGB gene expression in human trophoblastic (blastocysts, n = 6; normal/failed pregnancy, n = 51) and non-malignant non-trophoblastic tissues (15 different tissue types, samples n = 241), by real-time RT-PCR. We showed a wide transcriptional window of CGB genes in normal pregnancy, a significant reduction in recurrent miscarriages, and a high expression (especially CGB1/CGB2) in ectopic and molar pregnancies. Expression was several orders of magnitude lower in the non-placental tissues, with the highest CGB levels being seen in testis, prostate, thymus, skeletal muscle and lung samples. The contribution of CGB1/CGB2 to the summarized expression of six CGB genes was not proportional to their gene dosage: 1/1000 to 1/10,000. An interesting exception was the testis exhibiting a much higher CGB1/CGB2 to total CGB mRNA ratio of approximately one-third, corresponding to gene dosage. In conclusion, the expressional profile of CGB genes, activated already in blastocyst stage, is associated with the status of pregnancy. The presence of CGB transcripts in testes, and in particular CGB1/CGB2 transcripts, may indicate a role in male reproductive tract.

Androgen receptor gene haplotype is associated with male infertility.

The purpose of the current study was to evaluate the importance of androgen receptor (AR) gene haplotypes and polymorphic CAG/GGN microsatellites in the aetiology of male infertility. We genotyped six haplotype-tagging single nucleotide polymorphisms and CAG/GGN microsatellites of the AR gene in 112 infertile and 212 control Estonian men. A total of 13 AR haplotypes (HAP1-13) were identified, among which HAP4 was found to confer increased risk for male infertility (OR = 5.15, 95% CI = 1.75-15.15, p = 0.003). However, infertile patients and controls had similar lengths and distributions of both AR CAG (mean +/- SD number of repeats 21.1 +/- 2.5 vs. 21.2 +/- 2.3, respectively) and GGN (mean +/- SD number of repeats 22.5 +/- 1.5 vs. 22.4 +/- 1.9, respectively) repeats. In addition, HAP2 was associated with more CAG repeats (r = 1.17, p = 0.033) and HAP3 with fewer CAG repeats (r = -2.93, p < 0.001) than the major haplotype HAP1. HAP3 and HAP4 were associated with more GGN repeats (r = 1.35, p = 0.001 and r = 1.36, p = 0.002, respectively) than HAP1. In conclusion, our results implicated the AR-HAP4 gene haplotype in increased risk for male infertility, while no association was found between AR CAG/GGN microsatellites and impaired spermatogenesis.

Genetic predictors of controlled ovarian hyperstimulation: where do we stand today?

BACKGROUND: Nowadays, the use of IVF has improved the prospects of infertility treatment. The expected outcome of IVF depends greatly on the effectiveness of controlled ovarian hyperstimulation (COH), where exogenous gonadotrophins are used to induce folliculogenesis. The response to stimulation varies substantially among women and is difficult to predict. Several predictive markers of COH outcome have been proposed (e.g. maternal age and ovarian reserve), but the search for optimal predictors is ongoing. Pharmacogenetic studies demonstrate the effects of individual genetic variability on COH outcome and the potential for customizing therapy based on the patient's genome. METHODS: MEDLINE, EMBASE, DARE, CINAHL and the Cochrane Library, and references from relevant articles were investigated up to February 2011 regarding any common genetic variation and COH/IVF outcome. RESULTS: Several polymorphisms in genes involved in FSH signalling, estrogen biosynthesis, folliculogenesis, folate metabolism and other aspects influence the response to exogenous gonadotrophin administration, resulting in differences in COH and IVF outcomes. Nevertheless, the most studied polymorphism FSHR Asn680Ser is practically the only genetic marker, together with ESR1 PvuII T/C, that could be applied in clinical tests. CONCLUSIONS: Although data are accumulating with evidence suggesting that the ovarian response to COH is mediated by various polymorphisms, the optimal biomarkers and the efficacy of the tests still remain to be evaluated.

Cytogenetic and molecular characterization of the derivative Y chromosome: a case study of an azoospermic patient.

The success of infertility treatment depends on the underlying cause and severity of the infertility problem. The current report addresses the complex genotype-phenotype interactions in an azoospermic man. Cytogenetic, molecular cytogenetic and molecular genetic studies indicated the derivative monocentric Y chromosome with duplication of Yp11 (including SRY gene) and partial deletion of Yq11 (including azoospermia factor - AZFb-c regions) as the most probable cause of the severe testicular failure. Our study emphasizes the importance of detailed genetic analysis in male infertility evaluation and helps to estimate the outcome of infertility treatment.