Chronic endometritis and the endometrial microbiota: implications for reproductive success in patients with recurrent implantation failure.

BACKGROUND: Chronic endometritis (CE) is associated with poor reproductive outcomes, yet the role of endometrial microbiota in patients with recurrent implantation failure (RIF) and CE remains unclear. This study aims to characterize endometrial microbiota in RIF patients with CE and assess its implications for reproductive outcomes. METHODS: In this prospective study, we enrolled RIF patients both with and without CE. Endometrial and cervical samples were collected for 16 S rRNA gene sequencing. Microbiota composition was compared between groups using diversity indices, phylum, and genus-level analysis. Canonical correlation analysis (CCA) and Spearman's correlation coefficients were used to assess relationships between CE, reproductive outcomes, and microbiota. Predictive functional profiling was performed to evaluate metabolic pathways associated with CE. RESULTS: Endometrial microbiota in CE patients exhibited greater diversity and evenness compared to non-CE patients. Principal coordinates analysis (PCoA) revealed distinct clustering between CE and non-CE groups. Linear discriminant analysis (LDA) identified Proteobacteria, Aminicenantales, and Chloroflexaceae as characteristic of CE, while Lactobacillus, Acinetobacter, Herbaspirillum, Ralstonia, Shewanela, and Micrococcaceae were associated with non-CE. CCA demonstrated associations between CE, adverse reproductive outcomes, and specific bacterial taxa. Microbial metabolic pathways significantly differed between CE and non-CE groups, with enrichment in pathways related to cofactors, vitamins, secondary metabolites, and the immune system in CE patients. CONCLUSION: RIF patients with CE exhibit distinct endometrial microbiota compositions associated with adverse reproductive outcomes. The increased microbial diversity and altered metabolic pathways in CE suggest a potential correlation with reproductive outcomes, although further studies are necessary to elucidate the causal relationship between microbiota alterations and fertility. Modulating the endometrial microbiome may represent a novel therapeutic strategy to improve IVF outcomes in patients with CE.

Single-Cell RNA Transcriptome of the Human Endometrium Reveals Epithelial Characterizations Associated with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) remains a complex and poorly characterized disorder despite significant advancements in assisted reproductive technology. This study utilizes single-cell transcriptome sequencing (scRNA-seq) to characterize the mid-secretory endometrium of RIF patients. Stromal fibroblast-enriched and epithelium-enriched populations are collected using a two-step dissociation process. After quality control, 25,315 individual cells from 3 RIF patients are analyzed. The analysis identifies 12 distinct cell types, including 6 subtypes of epithelial cells. Significantly, the study reveals the replacement of glandular epithelia with MAP2K6+ EPCAMDIM epithelia in the endometrial glands of RIF patients. Furthermore, the study demonstrates that endometrial gland organoids derived from RIF patients exhibit diminished responses to sex steroids compared to the controls. Single-cell regulatory network inference and clustering (SCENIC) analysis identifies cell-specific cis-regulatory elements and constructed regulatory networks in both groups, showing alterations gene-regulatory networks in RIF patients. Cell-cell communication analysis distinguishes intercellular communication between the two groups, shedding light on disrupted cellular interactions associated with RIF. In summary, these findings provide valuable insights into the cellular and molecular mechanisms underlying RIF, highlighting the roles of epithelial cells in the implantation process.

Metabolomics analysis of serum metabolites during endometrial transformation: association with recurrent implantation failure in hormonal replacement therapy-frozen embryo transfers cycles.

PURPOSE: The purpose of this study was to investigate alterations in serum metabolites during endometrial transformation and possible associations with recurrent implantation failure (RIF) in hormonal replacement therapy (HRT)-frozen embryo transfer (FET) cycles. METHODS: We performed a prospective study involving 100 patients scheduled for HRT-FET cycles during January 2022 to April 2022. Blood serum samples were collected on the day of progesterone administration (dPA) and on the third day of progesterone administration (d3PA). Gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify and quantify serum metabolites. A nested case-control study including 19 RIF patients and 19 matching controls was conducted to explore the predictive value of serum metabolites for RIF. Partial least squares discriminant analysis (PLS-DA) and receiver operating characteristic (ROC) curve analysis were performed to establish prediction models. MAIN RESULTS: We identified 105 serum metabolites, with 76 of them exhibiting significant alterations during the initial 3 days of endometrial transformation. Metabolites involved in amino acid metabolism and tricarboxylic acid (TCA) cycle showed lower levels during endometrial transformation. In the nested case-control study, the prediction model based on the ratio of serum metabolites between d3PA and dPA showed the highest area under the ROC curve (AUC), accuracy, and R2 and Q2 values. Eight metabolites, including indol-3-propionic acid, beta-alanine, myristoleic acid, malic acid, indole, DL-isocitric acid, proline, and itaconic acid, exhibited high predictive values for RIF. CONCLUSION: This study demonstrates alterations in serum metabolites during endometrial transformation, particularly in amino acid metabolism and TCA cycle. The identified metabolites, especially indol-3-propionic acid and malic acid, show potential as predictive markers for RIF. These findings contribute to a better understanding of the metabolic changes associated with endometrial receptivity and provide insights for the development of personalized approaches to improve implantation outcomes in FET cycles.

Effect of chronological age of patients with diminished ovarian reserve on in vitro fertilization outcome.

The aim of this study was to evaluate the effect of chronological age on the in vitro fertilization (IVF) outcome in patients with diminished ovarian reserve (DOR). Four hundred and forty-nine women with DOR who underwent IVF cycles were enrolled in the study. There were only 296 patients who obtained available embryos. The patients with no available embryos had a significantly lower antral follicle count (AFC) and higher basal follicle-stimulating hormone (FSH) concentrations than those of women with available embryos, but chronological age in the two groups was comparable. However, patients aged >40 obtained a significantly lower ongoing pregnancy rate (OPR) than patients aged 35 - 40 or <35 (6.38% versus 26.15% versus 28.17%, respectively). Multivariate analysis also showed that chronological age was the only parameter associated with clinical results. It implied that patients with DOR still have reasonable chances of achieving a pregnancy, but their prognosis is significantly affected by chronological age.Impact statementWhat is already known on this subject? Diminished ovarian reserve DOR is a disappointing issue in reproductive medicine. Ovarian reserve, which represents ovarian biological age, is closely related to chronological age. However, ovarian biological age does not always match chronological age. Some studies suggest that biological age is more important than chronological age in predicting the outcome of in vitro fertilization (IVF). However, these conclusions are controversial.What do the results of this study add? We found DOR patients with no available embryos had significantly lower antral follicle count (AFC) and higher follicle-stimulating hormone (FSH) concentrations than that of patients with available embryos, but chronological age in the two groups was comparable. However, for patients with available embryos, chronological age is the only parameter associated with clinical results. For women aged >40 with DOR, chronological age was significantly negatively associated with clinical results.What are the implications of these findings for clinical practice and/or further research? Patients with DOR can obtain available embryos and still have a reasonable chance of becoming pregnant, but their prognosis is greatly affected by chronological age. Therefore, patients with DOR should seek medical help for pregnancy as soon as possible. When DOR patients over the age of 40 plan IVF treatment, the cost-effectiveness of healthcare should be considered.

Urinary vitamin D-binding protein as a marker of ovarian reserve.

BACKGROUND: Ovarian reserve reflects the quality and quantity of available oocytes and has become an indispensable measure for the better understanding of reproductive potential. Proteomic approaches are especially helpful in discerning differential protein expression patterns associated with normal and diseased states and, thus, proteomic analyses are increasingly used to identify clinically useful biomarkers. The aim of this study was to investigate proteins secreted in the urine of patients with different ovarian reserve by proteomic techniques to identify potential markers for assessing ovarian reserve. METHODS: Urine samples were obtained from patients with polycystic ovary syndrome (PCOS) and diminished ovarian reserve (DOR), and from normal control (NC)participants. We used isobaric tags for relative and absolute quantification (iTRAQ) technology combined with mass spectrometry analysis to identify candidate urinary proteins in the three groups. The selected proteins were confirmed using western blot analysis and enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of the selected proteins was assessed using receiver operating characteristic analysis. RESULTS: When Compared with NC samples, 285 differentially expressed proteins (DEPs) were identified in the DOR samples and 372 in the PCOS samples. By analyzing the intersection of the two groups of DEPs, we found 26 proteins with different expression trends in the DOR and PCOS groups. Vitamin D-binding protein (VDBP) was the key protein for the protein-protein interaction network. ELISA quantification of urinary VDBP revealed the highest levels in the PCOS group, followed by the NC group and the lowest levels in the DOR group (115.90 ± 26.02, 81.86 ± 23.92 and 52.84 ± 21.37 ng/ml, respectively; P < 0.05). As a diagnostic marker, VDBP had a sensitivity of 67.4% and a specificity of 91.8% for DOR, and a sensitivity of 93.8% and a specificity of 77.6% for PCOS. CONCLUSIONS: Urinary VDBP is closely associated with ovarian reserve and can be considered as a novel noninvasive biomarker of ovarian reserve. However, studies including large sample sizes are needed to validate these results.

Transplantation of Human Amnion Epithelial Cells Improves Endometrial Regeneration in Rat Model of Intrauterine Adhesions.

Intrauterine adhesions (IUAs) are characterized by the injury of endometrium due to curettage and/or endometritis. The loss of functional endometrium in uterine cavity usually results in hypomenorrhea, amenorrhea, infertility, and/or recurrent pregnancy loss. Recently, stem cell transplantation has been applied to promote the endometrial regeneration. Human amnion epithelial cells (hAECs) have been shown to have stem cell characteristics. In this study, we found that PKH26-labeled hAECs were mainly distributed in the basal layer of endometrium after transplantation, and hAEC transplantation, including uterine injection and tail vein injection, could increase pregnancy rate and the number of embryos in rat model of IUAs. Moreover, hAEC transplantation was demonstrated to increase the endometrial thickness, promote the proliferation of glands and blood vessels, and decrease fibrotic areas in the endometrium. The immunohistochemical and quantitative polymerase chain reaction analysis showed the upregulated expression of growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) after hAEC transplantation; and the downregulated expression of collagen type I alpha 1 (COL1A1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-ß (TGF-ß), all of which are associated with the extracellular matrix (ECM) deposition after hAEC transplantation. The mRNA sequencing indicated that platelet-derived growth factor-C (PDGF-C), thrombospondin-1 (THBS1), connective tissue growth factor (CTGF), Wnt5a, and Snai2 were significantly modulated in treatment groups. These results indicate that hAEC transplantation promotes endometrial regeneration and the restoration of fertility in rat model of IUAs.

Effect of human amniotic epithelial cells on ovarian function, fertility and ovarian reserve in primary ovarian insufficiency rats and analysis of underlying mechanisms by mRNA sequencing.

Human amniotic epithelial cells (hAECs) show similar features to stem cells and have low immunogenicity. This study aims to investigate the therapeutic effect of hAEC transplantation on cyclophosphamide-induced primary ovarian insufficiency (POI) rats and evaluate the underlying mechanisms by mRNA sequencing of ovarian samples. Notably, hAECs mainly located in the interstitial area of the ovaries rather than follicles. hAEC transplantation led to a slight increase in body and ovary weight, normalized irregular estrous cycles, decreased serum follicle stimulating hormone (FSH) and increased anti-Mullerian hormone (AMH) level and restored follicle pools in POI rats. Ovarian expression of AMH, follicle stimulating hormone receptor (FSHR) and klotho in POI rats was also significantly upregulated following hAEC transplantation. Fetus number was higher in the hAEC transplantation group than the POI group. The mRNA sequencing results showed that hAEC transplantation led to the upregulation of several angiogenesis and inflammation molecules including interferon regulatory factor 7 (IRF7), Mx dynamin-like GTPase 1 (Mx1), vascular endothelial growth factor receptor (VEGFR)1 and VEGFR2. Moreover, hAEC therapy had an effect on ribosomes, protein digestion, protein absorption, neuroactive ligand-receptor interaction, cAMP signaling pathway and steroid biosynthesis pathways. The expression of several steroid biosynthesis proteins was significantly upregulated as measured by quantitative real-time polymerase chain reaction (RT-qPCR), immunohistochemical staining and Western blot analysis. In summary, hAECs can significantly restore ovarian function, and improve both ovarian reserve and fertility. This may be due to the paracrine effect of hAECs in regulating steroid biosynthesis, modulating follicle development from initiation to ovulation, promoting angiogenesis and reducing inflammation.

Protein Expression Profile in IVF Follicular Fluid and Pregnancy Outcome Analysis in Euthyroid Women with Thyroid Autoimmunity.

The objective of this study is to investigate the influence of the thyroid autoantibodies on the protein expression in follicular fluid and the clinical outcome of assisted reproductive technology. A total of 602 patients treated for infertility were screened; 49 euthyroid women who were positive for thyroid autoantibodies and 63 negative controls were recruited. Follicular fluid samples were analyzed using proteomics. Validation of target proteins in follicular fluid was performed by using parallel reaction monitoring. Differentially expressed proteins in follicular fluid, clinical pregnancy rate, abortion rate, and live-birth rate were analyzed. Clinical pregnancy rates and take-home baby rates in the thyroid autoimmunity (TAI) group were less than in the control group, but abortion rates in the TAI group were higher than in the control group (all P < 0.005). A total of 49 proteins were differentially expressed in the TAI-positive group. In Gene Ontology secondary annotations of all the proteins identified, five types of proteins were associated with the reproductive process. Among 11 proteins quantitatively identified by parallel reaction monitoring, angiotensinogen and fetuin-B were associated with reproduction. These differentially expressed proteins identified in this study involved multiple pathways according to the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Our study provides evidence that some differentially expressed proteins between TAI-positive women and controls were associated with the reproductive process and closely related to important physiologic effects, which could partially explain the underlying mechanism link between TAI and the adverse outcomes of assisted reproductive technology.

Adverse effect of paternal hepatitis B virus infection on clinical pregnancy after frozen-thawed embryo transfer.

PURPOSE: This study aimed to determine the effect of paternal hepatitis B virus (HBV) infection on reproductive outcomes of couples undergoing frozen-thawed embryo transfer (FET). METHODS: This retrospective cohort study included FET cycles performed between January 2014 and March 2017 in couples with a hepatitis B surface antigen (HBsAg)-positive male partner and an HBsAg-negative female partner, which was categorized as HBsAg group. The FET cycles underwent by couples with both HBsAg-negative partners were randomly selected as controls. The primary outcome was clinical pregnancy. RESULTS: A total of 117 FET cycles, comprising 39 in the HBsAg group and 78 in the control group, were included. Couples with HBsAg-positive male partners had significantly lower clinical pregnancy rate (17.9 vs 41.0%, P = 0.013), lower implantation rate (11.1 vs 24.5%, P = 0.014), and lower live birth rate (12.8 vs 30.8%, P = 0.034) compared with the control group. Moreover, the multivariate logistic regression analysis showed that paternal HBV infection was negatively associated with clinical pregnancy (odds ratio = 0.297, 95% confidence interval 0.108-0.817, P = 0.019). The miscarriage rate was not significantly different between the two groups (28.6 vs 25.0%, P = 1.000). CONCLUSIONS: Paternal HBV infection resulted in a lower frequency of clinical pregnancy after FET, a difference that was probably attributed to a detrimental effect of HBV on the ability of embryos to survive freezing and thawing.

Assisted Hatching in Couples with Advanced Maternal Age: A Systematic Review and Meta-analysis.

This systematic review aimed to evaluate the efficacy and safety of assisted hatching (AH) performed in couples with advanced maternal age. We searched for randomized controlled trials (RCTs) in electronic databases, including MEDLINE, EMBASE and CENTRAL (from inception to January 2018); in addition, we hand-searched the reference lists of included studies and similar reviews. We included RCTs comparing AH versus no treatment (control). The meta-analysis was performed by RevMan 5.3 software. The search retrieved 943 records and 8 RCTs were included, comprising 870 cycles (n=440 for AH, and n=430 for control). There was no significant difference in the rates of live birth (RR 0.88, 95% CI 0.65 to 1.18, 3 RCTs, n=427, I2=0%), clinical pregnancy (RR 1.00, 95% CI 0.83 to 1.19, 8 RCTs, n=870, I2=22%), implantation (RR 1.07, 95% CI 0.83 to 1.39, 4 RCTs, n=1359, I2=0%), miscarriage (RR 1.13, 95% CI 0.66 to 1.94, 2 RCTs, n=116, I2=0%) and multiple pregnancy (RR 0.89, 95% CI 0.31 to 2.52, 1 RCT, n=97, I2=not applicable) between the treatment group and control group. No reasonable conclusions could be drawn regarding reproductive outcomes after AH in patients with advanced maternal age due to the small sample pooled in meta-analyses. Studies of high methodological quality and with adequate power are necessary to further investigate the value of AH in assisted conception of those patients.

[IMSI versus ICSI for male factor infertility: A meta-analysis].

OBJECTIVE: To evaluate the efficacy and safety of intracytoplasmic morphologically selected sperm injection (IMSI) versus intracytoplasmic sperm injection (ICSI) in in vitro fertilization (IVF) for couples with male factor infertility. METHODS: Using the Cochrane system evaluation method, we searched MEDLINE, EMBASE, CENTRAL, ClinicalTrials.gov, and SinoMed and manually searched the reference lists of the included studies and relevant reviews for randomized controlled trials (RCT) comparing ICSI and IMSI published from 1992 to July 2017. We performed a meta-analysis on the included literature with the RevMan 5.3 software and subgroup analyses due to the prominent clinical heterogeneity of the patients. RESULTS: Of the 280 articles retrieved, 8 RCTs were included, involving 1 741 IVF cycles (842 cycles of IMSI versus 899 cycles of ICSI). There was no evidence for any significant difference between IMSI and ICSI in the live birth rate in the subgroup of infertility induced by pure male factors (RR = 1.31, 95% CI: 0.68－2.51; very low quality evidence from 1 RCT with 77 cycles) but an association of IMSI with an increased clinical pregnancy rate (RR = 1.46, 95% CI: 1.02－2.07; low quality evidence from 4 RCTs with 813 cycles), nor was there any evidence for that in the live birth rate (RR = 0.88, 95% CI: 0.60－1.31; low quality evidence from 1 RCT with 255 cycles) or clinical pregnancy rate (RR = 1.03, 95% CI: 0.86－1.23; moderate quality evidence from 3 RCTs with 851 cycles) in the subgroup of infertility caused by accompanying male factors. CONCLUSIONS: The evidence is of low quality for the association of IMSI with an increased rate of clinical pregnancy and is not sufficient to support the routine use of IMSI in IVF for male factor infertility.

Evaluation of the spermicidal and contraceptive activity of Platycodin D, a Saponin from Platycodon grandiflorum.

BACKGROUND: The extract of Platycodon grandiflorum has been reported to have effective spermicidal activity. This study was designed to evaluate the spermicidal and contraceptive activity, as well as the safety, of Platycodin D (PD), a major saponin in Platycodon grandiflorum. METHODS: Using the computer-aided sperm analysis (CASA) test criteria, the sperm-immobilizing activity of PD was studied using highly motile human sperm. The sperm viability was assessed by fluorescent staining using SYBR-14 (living sperm) and propidium iodide (dead sperm). The sperm membrane integrity was assessed by evaluating the hypo-osmotic swelling (HOS) and examinations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The in vivo contraceptive efficacy was evaluated in rats using post-intrauterine PD application. The comet assay was employed to determine whether PD caused DNA damage in the sperm. Vaginal biopsies were also performed to determine whether the PD gel induced vaginal inflammation. RESULTS: A dose-dependent effect of PD on the sperm motility and viability was observed. The maximum spermicidal effect was observed with a 0.25 mM concentration of PD. More than 70% of the PD-treated sperm lost their HOS responsiveness at a concentration of 0.20 mM PD, indicating that PD caused injury to the sperm plasma membrane. TEM and SEM revealed significant damage to both the head and tail membranes of the sperm. PD decreased the fertility to zero in rats, was non-DNA damaging and was not harmful to the vaginal tissue in the rats. CONCLUSION: PD has significant spermicidal activity that should be explored in further studies.

Fallopian tube occlusion with a shape memory polymer device: evaluation in a rabbit model.

BACKGROUND: The present study evaluates the feasibility of a shape memory polymer (SMP) device for fallopian tube occlusion in rabbits. STUDY DESIGN: The SMP contraceptive device is made of poly(dl-lactic acid)-based poly(urethane urea) SMP in the shape of a spiral cylinder that was 10 mm long and had a diameter of 2.6 mm. Using this device, bilateral transuterine fallopian tube occlusions were performed in 78 New Zealand white female rabbits. Forty-eight female rabbits (group 1) were chosen as the experimental group and were implanted with the SMP devices. The remaining 30 female rabbits (group 2) served as the control group, which only received an incision in the abdomen but no SMP device. Follow-up consisted of hysterosalpingography, histologic evaluation and contraceptive effect. In addition, the shape memory behavior and in vivo degradation characterization of the SMP device were observed in this study. RESULTS: Under heat (37 °C) stimulation, the temporary shape SMP device returned to its permanent shape within 60 s. The average weight loss percentage of SMP devices was 7.0% at 2 weeks and 72.5% at 12 weeks. The inflammatory reactions caused by SMP devices were aseptic and nonspecific at 2 and 12 weeks, respectively. The SMP device boundaries and the surrounding tissues were obscured by fiber hyperplasia in 11/12 tubes at 24 weeks. Hysterosalpingography showed an occluded fallopian tube of Group 1 in 6/6 rabbits at 12 weeks and 6/6 rabbits at 24 weeks. No pregnancy was found in all 18 rabbits of group 1 (contraceptive rate of 100%); all 20 rabbits in the control group were pregnant. CONCLUSION: Biodegradable and biocompatible SMP devices could provide reliable, instant and permanent tubal occlusion.

[Regulation of epidermal growth factor, thyrotropin releasing hormone and dexamethasone on the expression of fetal lung SP mRNAs in rabbits].

OBJECTIVE: To compare the effect of epidermal growth factor (EGF), thyrotropin releasing hormone (TRH) and dexamethasone (Dex) on the expression of surfactant protein (SP)-A, SP-B and SP-C mRNA in fetal rabbit lungs. METHODS: Sixty-four Japanese white pregnant rabbits, put into four different groups (16 rabbits each), were given EGF (EGF group), TRH (TRH group), Dex (Dex group) or normal saline (control group) intravenously from gestational day 22(nd) (5 rabbits in each group) or 24(th) (11 rabbits in each group) for three days before killing. The levels of SP-A, SP-B and SP-C mRNAs in the fetal rabbit lungs were detected by reverse transcription polymerase chain reaction (RT-PCR) and Southern blotting. RESULTS: In the 27(th) gestational day, SP-B mRNA levels in the fetal rabbit lungs in all treated groups were found significantly higher than those in the control group (P < 0.05). In the 25(th) day, there had a higher expression of SP-A and SP-C mRNAs in the fetal lungs in all groups, but SP-B mRNA could be tested only in the Dex group. SP-B mRNA level was lower than that of SP-A mRNA and SP-C mRNA in the same fetal lungs in the 27(th) gestationed day. CONCLUSION: The time of detectable SP-B mRNA in rabbit fetal lungs appeared later than that of the SP-A and SP-C mRNAs. The expression of SP-B mRNA is lower in comparison with those of SP-A mRNA and SP-C mRNA in the same samples. Prenatal maternal treatment with intravenously Dex, EGF and TRH could increase the expression of SP-B mRNA in fetal rabbit lungs, and the effect of Dex may be more significant at earlier stage of gestation.