Sirtuin expression in the gravidar endometrium during an undeveloped pregnancy

BACKGROUND: Early reproductive loss is a complex problem, the solution of which requires a systematic approach to the study of the morphogenesis of gravid transformation. Violations of the morphofunctional characteristics of the endometrium are associated with chronic endometritis, which entails implantation pathology and the development of pregnancy, while formulating indications for the use of IVF methods. Chronic endometritis disrupts the differentiation of stromal cells and intermediate filaments with subsequent pathology of decidualization and gravid transformation. AIM: The aim of this study was to evaluate vimentin expression in decidual cells of the gravid endometrium in an undeveloped first-trimester pregnancy after IVF, depending on its transformation in patients with chronic endometritis. MATERIALS AND METHODS: Histological and immunohistochemical studies were performed on 40 samples of abortion material with normal chorionic villi karyotype from a non-developing pregnancy after IVF in patients with chronic endometritis and 15 samples of abortion material from a progressive pregnancy, surgically terminated at the woman’s request at similar terms. Two groups were formed — I group with a full-fledged gravid transformation in a non-developing pregnancy after IVF (n = 20) and II group with an incomplete gravid transformation in a non-developing pregnancy after IVF (n = 20). Histological examination was performed according to the standard procedure. Immunohistochemical study was performed using a one-step protocol with antigen unmasking. Primary monoclonal antibodies to vimentin (Clon V9, Diagnostic BioSystems Inc., Spain) were used at a 1 : 1000 dilution. Vimentin expression was evaluated in the compact layer of the gravid endometrium. RESULTS: A full-fledged gravid transformation of the endometrial stroma in the abortion material in study group I and the control group was represented by mature deciduocytes of the epithelioid type, round or polygonal in shape, with a light vesicular nucleus. With an incomplete gravid transformation, the compact layer of the gravid endometrium was represented by fibroblasts with varying degrees of cytoplasmic development, from mild changes to the formation of pre-decidual and immature decidual cells. A decrease in vimentin expression was verified in the gravid endometrium after IVF compared to the control group. In case of an incomplete gravid transformation, a decrease in vimentin expression was also verified compared to the control group and study group I. CONCLUSIONS: The presence of chronic endometritis in patients with infertility and chronic endometritis not only causes pathology of the original endometrium, but also disrupts decidualization, leading to reproductive loss with normal embryo karyotype.

Should patients be screened for chronic endometritis before assisted reproductive technology?

Consistent evidence on the detrimental role of severe chronic endometritis on in vitro fertilization outcome and the reproductive improvement after antibiotic therapy: on the other hand, mild chronic endometritis appears a more intricate matter

Study question Can we develop a real-time diagnostic tool for chronic endometritis (CE) by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to evaluate biopsies obtained during hysteroscopy? Summary answer A discrimination model based on the absorbance data was developed by machine learning techniques, differentiating between positive and negative CE histopathology with 97% accuracy. What is known already CE is diagnosed in approximately 15% of infertile women who undergo in vitro fertilization (IVF), in 42% of women with recurrent implantation failure (RIF), and in 57.8% of women with RPL. Diagnosis is done by endometrial biopsy, and the presence of plasma cells in the endometrial stroma is the generally accepted histological diagnostic criterion. However, the histological detection of CE is time-consuming and difficult. ATR-FTIR spectroscopy is a non-destructive method that can provide valuable information on biochemical changes that occur during pathological processes, such as inflammation and cancer. Study design, size, duration We performed a prospective study in which fresh biopsies of endometrium were obtained during standard hysteroscopies. Each biopsy was examined by the spectrophotometer and afterward by histopathological analysis in which multiple myeloma oncogene 1 (MUM–1) staining for plasma cells, a marker of CE, was performed. We planned to investigate 80 samples to develop a discrimination model, and another 40 samples for validation of the model. The study was planned to last two years. Participants/materials, setting, methods Women that underwent hysteroscopy as a part of infertility evaluation were recruited. The hysteroscopies and the biopsy evaluation were performed at the same center. A cut-off of 8 MUM–1 positive cells per 10 high power fields (HPF) was set. We compared the spectroscopy analysis of the positive CE group (≥8) and the negative CE group (<8). Machine learning technique was utilized to build discrimination models. Data analysis was performed using Matlab and Unscrambler software packages. Main results and the role of chance We present preliminary results for our study. Forty-two women were recruited from January 2020 until November 2020. Of the 42 measured spectra, three were discarded due to high measurement noise. Of the 39 biopsies, 33 had MUM–1<8 (CE negative group) and 6 had MUM–1≥8 (CE positive group). Measured spectra of tissue smears from CE negative and positive groups differed from each other in the spectral range of 850–990 [cm–1] (p < 0.05). This wavenumber can be associated with the C-H in-plane bend in the alkene group (CnH2n). A discriminant model was developed between the groups using the Principal Component Analysis and Linear Discriminant Analysis techniques. The accuracy obtained by the model was 97%. We divided the 39 hysteroscopies based on the CE signs into 2 groups: “Negative hysteroscopic-CE” and “Positive hysteroscopic-CE”. Positive hysteroscopic signs were micropolyps, strawberry pattern, hyperemia, punctuation, or pale endometrium. Twenty-three samples were taken in the Negative group and 16 samples were taken in the Positive group. However, measured spectra of tissue smears from negative and positive hysteroscopy groups were not significantly different. The correlation coefficient between hysteroscopy groups and MUM–1 score was r = 0.29, meaning that the characteristic signs of CE in hysteroscopy were not correlated to the histopathology. Limitations, reasons for caution First, these are preliminary results and we need to investigate more samples to validate our model. Second, diagnostic criteria for CE are diverse in the literature and we chose 8 MUM–1 positive cells in 10 HPF, a criterion which may not be accepted by all experts in the field. Wider implications of the findings: ATR-FTIR spectroscopy is highly sensitive to molecular changes and has been utilized as a diagnostic tool in a variety of clinical studies. While histopathological results take about two weeks, ATR-FTIR spectroscopy might give us the possibility to diagnose CE in real-time, allowing an immediate initiation of the appropriate treatment. Trial registration number ClinicalTrials.gov Identifier: NCT04197167

A review of current knowledge on the pathophysiology, diagnostic and treatment options for chronic endometritis in infertile women. One of the major causes of failed in vitro fertilization (IVF) is undiagnosed intrauterine pathologies, including chronic inflammation of the uterine mucosa - chronic endometritis. However, some authors relativize the negative impact of chronic endometritis on reproductive outcomes. The etiopathogenesis of chronic endometritis is due to qualitative and quantitative changes in the endometrial microbiome with abnormal multiplication of microorganisms naturally occurring in the uterine cavity or vagina. There is no uniform consensus on the most common pathogen causing chronic endometritis. It is characterized by infiltration of plasma cells into the endometrial stroma outside the menstrual cycle, accompanied by hyperaemia and endometrial oedema. Clinical symptoms are very mild or absent. The diagnosis of chronic endometritis is often difficult because there is no specific clinical or laboratory diagnostic method. The following investigative options are commonly used for the diagnosis of chronic endometritis: diagnostic hysteroscopy, histopathological examination of the endometrium including CD 138 immunohistochemistry and culture from the uterine cavity. However, standardised international hysteroscopic and histopathological criteria for accurate diagnosis of chronic endometritis are still lacking. Empirically administered antibiotic therapy improves the success rate of pregnancy and delivery of a viable foetus in infertile patients with proven chronic endometritis. In addition to reviewing the current knowledge of chronic endometritis, this article discusses the importance of hysteroscopy in the diagnostic process. Chronic endometritis is often a clinically silent disease with negative impact on reproduction in infertile women. Although there are still many unresolved issues, the introduction of hysteroscopy into the diagnostic process is important for clinical practice; however, hysteroscopy even in combination with histological examination of the endometrium, often does not allow an unequivocal diagnosis of chronic endometritis. Further prospective randomised studies in a selected group of women with proven chronic endometritis and repeated failure to implant proven euploid embryos should refine this knowledge.

Objective. To evaluate serum concentrations of leptin and adiponectin and their association with chronic endometritis (CE) in reproductive-aged women. Patients and methods. The study included 198 reproductive-aged women who were recruited during general health checks. All patients underwent anthropometric (body mass index (BMI), body fat percentage) and instrumental (pelvic ultrasound, endometrial pipelle biopsy with CD138 immunohistochemistry) examinations, and serum leptin and adiponectin levels were determined. Non-parametric methods and logistic regression were used for statistical analysis. Results. The results of the examination of 121 women were analyzed, of whom 20 were subsequently excluded due to missing data on BMI values, serum leptin concentrations, and CD138 expression levels in the endometrial stroma. Patients were divided into two groups: 60 healthy women (control group) and 41 women with CE, between whom statistically significant differences in BMI (p = 0.012), leptin (p = 0.002) and adiponectin (p = 0.002) levels were determined. Following patients’ division into subgroups according to BMI, women with CE were found to have statistically significantly higher adiponectin levels (p = 0.01) in the subgroup with normal weight and lower leptin levels (p = 0.001) in the subgroup with BMI ≥25 kg/m2 compared to that in the control group. Relatively low leptin levels were a risk factor for CE (OR: 0.903 [0.843; 0.969], p = 0.004) in overweight and obese women. Conclusion. A significant association between serum leptin levels and the presence of CE was determined in overweight and obese women, but not in normal weight women. Key words: adiponectin, inflammation, leptin, overweight, body mass index, obesity, chronic endometritis

BackgroundThe presence of chronic deciduitis (CD) was determined in patients diagnosed with or without chronic endometritis (CE) before pregnancy.ObjectiveTo study the effect of CE on decidua in cases of miscarriage.MethodsDecidual tissue was obtained from the patients who miscarried at the first pregnancy within a year after the diagnosis of the presence or absence of CE. The number and distribution pattern of plasma cells stained with CD138 in decidual tissue in 10 high-power fields (HPFs) was examined. The prevalence of CD diagnosed with four different grade; grade 0, no plasma cell in 10 HPFs, thus Non-CD;grade 1, rare single plasma cells; grade 2, rare clusters or more than 5 single cells total; and grade 3, many plasma cells with more than 5 clusters, were examined and compared between Non-CE and CE.ResultsThe incidence rate of CD of grade2 + 3 was significantly higher in CE than Non-CE (53.8%; 7/13 vs. 0%; 0/13, P < 0.01). Presence of clusters or a number of plasma cells in 10 HPFs of decidua showed a sensitivity of 53.8%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 68.4% for the diagnosis of CE.ConclusionPresence of clusters of plasma cells or five or more of plasma cells in decidua was found in more than half of CE, but not found in Non-CE. When CD with cluster or five or more of plasma cells is confirmed histologically in miscarriage decidual tissue, the presence of CE before the pregnancy should be suspected.

The aim is to identify the chronic endometritis (CE) incidence in recurrent implantation failure (RIF) patients undergoing in vitro fertilization (IVF) treatment and compare the IVF outcomes of RIF patients with CE following antibiotic therapy with RIF patients without CE. Another purpose is to compare the IVF outcomes of described RIF patients with patients undergoing the first cycle of IVF. In this retrospective cohort study, CE was diagnosed with CD-138 immunohistochemical staining. Among RIF patients, two groups were formed as group 1, including patients diagnosed with CE and treated by antibiotics (n=129), and group 2, including patients without CE (n=103). Patients with the first IVF cycle having similar infertility etiologies with RIF patients were reviewed as group 3 (n=932). CE was diagnosed in 55.6% of RIF patients. The number of oocytes retrieved was not different between groups. Implantation rates (IR) were similar after antibiotic treatment in RIF patients with or without CE. However, Group 3 had a higher IR (41.1%) than group 1 and 2 (23.1% and 30.1%, respectively) (p < 0.001). Clinical pregnancy (CPR) and live birth rates (LBR) were comparable between RIF groups. However, CPR and LBR were significantly higher in group 3 (48.6% and 40.5%) than group 1 (36.4% and 27.9%), and group 2 (37.9% and 30.1%) (p=0.007 and p=0.005, respectively). Unidentified endometrial factors except CE may also affect the implantation process, although CE is a frequent finding in patients with RIF. Reproductive outcomes may not be improved only with antibiotics in RIF patients with CE.

Background: Chronic endometritis (CE) is highly prevalent in patients with unexplained infertility. This study aimed to determine the associations between CE and infertility. Methods and Results: We performed a prospective study of 197 women with implantation failure and pregnancy loss after undergoing in vitro fertilization (IVF). The endometrium was examined with a hysteroscope, and a sample was taken for biopsy in areas suspected of having inflammation. Immunohistochemistry was performed for CD138 antibodies. Diagnostic criterion was more than 5 plasma cells per 10 high-power fields. Patients who were positive for CE were treated with antibiotics. Among the 197 patients in our study, 115(58.4%) were positive for CE. Regarding the success of CE treatment and pregnancy after treatment, among the 115 CD138-positive patients, 32 became pregnant without IVF after treatment, while another 43 had successful pregnancies and childbirth with IVF. Thus, with treated CE, a successful pregnancy and childbirth was detected in 65.2% of cases, including spontaneous pregnancies and IVF. Conclusion: Patients with infertility and those who have pregnancies lost in IVF have a high rate of CE. Failure to treat CE results in infertility and IVF failure. An efficient diagnosis and treatment of CE are important for a successful pregnancy.

Chronic endometritis: a common finding in good prognosis patients with failed implantation following in-vitro fertilization

This retrospective cohort study explored the morphological features of chronic endometritis (CE) and pregnancy outcomes during in vitro fertilization (IVF) in women with CE (429) at Yantai Yuhuangding Hospital between January 2017 and September 2018. The primary outcome was the clinical pregnancy rate (CPR). The women displayed haemorrhagic spots (175), hyperaemia (122), micropolyps (75), hyperaemia combined with micropolyps (49) and others (8). The CPR and live birth rate (LBR) were different among the hysteroscopic features of CE in fresh embryo transfer cycles (p = .002, p = .011). The miscarriage and premature birth rates were not significantly different among groups (p > .05). Hyperaemia (0.47 [95% CI, 0.29; 0.77]), micropolyps (0.40 [95% CI, 0.23; 0.72]), hyperaemia combined with micropolyps (0.35 [95% CI, 0.18; 0.69]) and others (0.36 [95% CI, 0.19; 0.69]) were associated with the CPR. In conclusion, the hysteroscopic features of CE are associated with IVF pregnancy outcomes, and there were differences in pregnancy outcomes with different CE hysteroscopic features. IMPACT STATEMENT What is already known on this subject? Chronic endometritis (CE) is associated with adverse pregnancy outcomes such as infertility, premature delivery and miscarriage. CE can reduce the success rate of pregnancy and even lead to obstetric and neonatal complications, and is an adverse factor for the success of in vitro fertilization (IVF). There are different types of CE, but their impact on IVF outcomes is unknown. What the results of this study add? The CPR and LBR were different among the hysteroscopic features of CE in fresh embryo transfer cycles (p = .002, p = .011). The miscarriage and premature birth rates were not significantly different among groups (p > .05). After adjustment, the multivariable analysis showed that hyperaemia (OR = 0.47, p = .002), micropolyps (OR = 0.40, p = .002), hyperaemia combined with micropolyps (OR = 0.35, p = .002) and others (OR = 0.36, p = .002) were associated with the CPR among patients with CE. What the implications are of these findings for clinical practice and/or further research? The hysteroscopic features of CE are associated with IVF pregnancy outcomes, and there were differences in pregnancy outcomes with different CE hysteroscopic features. Hence, women with repeated IVF failure should undergo hysteroscopy to examine for the presence of CE and its nature.

This systematic review and meta-analysis aims to evaluate the impact of chronic endometritis (CE) and its therapy on in vitro fertilization (IVF) outcome. Additionally, we aim to investigate whether various degrees of CE severity may exert a different effect on IVF outcome. Ongoing-pregnancy rate/live-birth-rate (OPR/LBR), clinical-pregnancy rate (CPR), and miscarriage rate (MR) were calculated. A total number of 4145 patients (from ten studies) were included. Women with CE had lower OPR/LBR (OR 1.97, p = 0.02) and CPR (OR 2.28, p = 0.002) compared to those without CE. CE cure increased OPR/LBR (OR 5.33, p < 0.0001) and CPR (OR 3.64, p = 0.0001). IVF outcome was comparable between women with cured CE and those without CE (OPR/LBR, CPR and MR: p = ns). Women with severe CE had lower OPR/LBR (OR 0.43, p = 0.003) and CPR (OR 0.40, p = 0.0007) compared to those mild CE. Mild CE showed no influence on the IVF outcome as compared to women without CE (OPR/LBR, CPR and MR: p = ns). Based on this data analysis, CE significantly reduces OPR/LBR and CPR in women undergoing IVF. Importantly, CE resolution after antibiotic therapy may improves IVF outcome, leading to similar OPR/LBR and CPR as compared to unaffected patients. The negative effects of CE on IVF outcome may be restricted to severe disease, whereas mild CE may have no influence on IVF success.

The objective of this prospective cohort study was to investigate the relationship between chronic endometritis (CE) and its treatment on pregnancy outcomes in patients undergoing frozen embryo transfers (FETs) from in vitro fertilization (IVF) with preimplantation genetic testing (PGT) who had previously failed ≥ 1 euploid FET. The diagnosis of CE was made using CD138 as a marker. This study occurred at a single, high volume academic based fertility center. 305 patients were included with a history of ≥ 1 failed euploid transfers. 59 patients underwent endometrial biopsies (EMB) prior to subsequent FET (Tested), 246 patients did not undergo EMB prior to subsequent transfer (Untested). Patients in the Tested group had an EMB prior to subsequent FET, whereas the patients in the Untested group did not have an EMB prior to next FET. Patients tested and found positive for CE were treated with antibiotics and underwent test-of-cure (TOC) EMBs prior to next FET to assess for adequate treatment. Patients without CE on EMB proceeded to next FET without antibiotic treatment. Main outcome measures included positive Beta-hCG (βhCG) on cycle day 28, implantation rate (IR), and subsequent ongoing pregnancy/live birth rates (OPR/LBR). Our results showed that 51% of patients (30/59) were diagnosed with CE and treated with antibiotics (Tested, CE treated); negative TOC biopsies were obtained prior to next FET. 49% of patients (29/59) tested negative for CE (Tested, No CE) and were not treated prior to next FET. Tested patients without CE (CE treated and No CE) had significantly higher incidences of positive βhCG, IR and OPR/LBR than patients who were not tested. Those who were biopsied and treated for CE had significantly higher OPR/LBR compared to patients who were biopsied and negative for CE (80% vs. 55%). We conclude that CE is common, with a prevalence of 51% in patients who have failed euploid FETs. Testing for CE may provide a benefit to patients, assuring higher incidences for positive βhCG, implantation, and subsequent ongoing/live births. CE is a treatable condition warranting investigation in patients with poor pregnancy outcomes.

Study question Could vaginal and endometrial microbiome by sequencing 16S rRNA be comparable to classic diagnostic methods or immunohistochemistry CD138 for diagnosis of chronic endometritis? Summary answer A characteristic endometrial and vaginal microbiome is present in patients with chronic endometritis. An abnormal vaginal microbiome correlates with the presence of chronic endometritis. What is known already Chronic endometritis is a disease characterized by persistent inflammation of the endometrial lining. Currently, histopathological evaluation by immunohistochemistry CD138 marker is most common diagnostic method for CE. Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of pathogenic microorganisms associated with CE. The main bacteria at vaginal and endometrial level belong to genus Lactobacillus, producers of lactic acid that allows maintaining acidic pH of vagina and acts as barrier against pathogens. Investigations on the effect of an abnormal endometrial and vaginal microbiome could improve assisted reproductive technologies. Study design, size, duration This is a observational pilot study (60 patients and 120 samples). The study population consists of patients attending to our fertility clinic for frozen euploid embryo transfer (FET) from May 2017 to May 2019. Preimplantation Genetic Testing of aneuploidy (PGT-A) was performed at blastocyst stage using Veriseq (Illumina). The inclusion criteria to be meet by patients were: age between 18 and 50 years, own or donated oocytes and use of ICSI. Participants/materials, setting, methods Cohort study with sixty patients undergoing assisted reproductive treatment (TRA) with their own or donated gametes and PGT-A Vaginal and endometrial samples were taken in the cycle prior to embryo transfer. The vaginal and endometrial microbiome was analyzed by mass sequencing of the V3V4 region of 16S rRNA. Bioinformatics analysis was performed using QIIME2 and MicrobiomeAnalyst packages. Alpha, beta diversity and taxonomic characterization were compared with positive and negative CD138 groups for chronic endometritis (CE). Main results and the role of chance Different bacterial communities were detected when vaginal and endometrial samples were analyzed in patients with and without endometritis diagnosed with CD138 immunohistochemistry. In patients with endometritis, a higher alpha diversity index tendency was found in vaginal samples (p = 0.15 for the Shannon index) and significant differences in endometrial samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between the groups established as per the diagnosis of endometritis. Vaginal and endometrial samples from women with endometritis showed a microbiome pattern not dominated by Lactobacillus spp. Relative abundance analysis identified the genera Ralstonia and Gardnerella in endometrial sample, and the genera Streptoccoccus and Ureaplasma in vaginal sample of patients diagnosed with CD138 for endometritis. Comparing endometrial and vaginal samples CD138 positive diagnosed for endometritis, alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p &amp;lt; 0.001) showed significant differences. Relative abundance identified the genera Lactobacillus (p = 3.76E-4), Ralstonia (p = 8.19E-4), Delftia (p = 0.004) and Anaerobacillus (p = 0.004) in these sample groups. Limitations, reasons for caution The main limitation of this study is the small sample size. Larger studies including a higher number of samples are needed to confirm the different microbiome pattern observed at the vaginal and endometrial levels in correlation with chronic endometritis. The microbiome pattern has not been analyzed after treatment of CE. Wider implications of the findings Our findings suggest the existence of a characteristic vaginal and endometrial microbiota in patients with chronic endometritis. Different genera and species were identified in patients with and without endometritis depending on whether the sample was endometrial or vaginal. An abnormal vaginal microbiome appears to be strongly correlated with chronic endometritis. Trial registration number Not Applicable

ObjectiveTo evaluate the endometrial histopathological profile of patients undergoing curettage and the association of the histopathological profile with the pregnancy outcome during the subsequent in-vitro fertilization (IVF) cycle.MethodsIn this retrospective cohort study, a total of 248 women with at least one failed attempt of IVF and who underwent curettage and a subsequent IVF were included. Demographic data, endometrial histopathological records, stimulation information, and pregnancy outcomes were collected and analyzed.ResultsThe histopathological analysis of endometrial tissues showed that 130 women (52.4%) had endometrial pathologies. Of these women, 103 (41.5%) had endometrial polyps, 22 (8.9%) had chronic endometritis, and five (2.0%) had both polyps and endometritis. No statistical difference was observed between the normal histopathology group and the abnormal histopathology group in the outcome of the subsequent IVF cycle. Subgroup analyses were performed to further characterize and compare women with normal histopathology and women with endometrial polyps (polyp subgroup) or chronic endometritis (endometritis subgroup). No statistical differences were found among the three groups in the rates of pregnancy (44.1% vs. 49.5% vs. 45.5%, P=0.72), biochemical pregnancy loss (13.5% vs. 15.7% vs. 20.0%, P=0.86), clinical pregnancy loss (25.0% vs. 31.4% vs. 30.0%, P=0.77), and live birth (27.1% vs. 26.2% vs. 22.7%, P=0.91) during the subsequent IVF cycle.ConclusionWomen with previously failed IVF and abnormal endometrial histopathology treated with curettage had the same outcome in the subsequent IVF cycle as women with normal endometrial histopathology.