Repeated Implantation Failure Research Articles

T REGULATORY, Th17, AND TREG/Th17 RATIO, IN PREGNANT WOMEN WITH RECURRENT PREGNANCY LOSSES AND NORMAL PREGNANT WOMEN

Dysregulated T regulatory cells and T helper (Th)-17 cells have been proposed as possible immune etiologies for recurrent pregnancy losses (RPL) and repeated implantation failures (RIF). In this study, we aim to investigate Th17, Treg cells, and the Treg/Th17 ratios during pregnancy in women with RPL (≥ 2 prior to 20 weeks) and normal pregnant women.

IMPACT OF MICROFLUID SPERM SORTING ON EMBRYO PLOIDY RATES IN COUPLES WITH HIGH SPERM DNA FRAGMENTATION?

Increased sperm DNA fragmentation (SDF) is one of the leading causes of male infertility. Conventional methods for sperm preparation have induced questioning of sperm recovery rates. The microfluidic sperm sorting (MSS) technique selects highly motile sperm with lower levels of SDF compared to density-gradient centrifugation (DGC) technique. This study aimed to compare MSS to DGC technique in couples with repeated implantation failure (RIF) and high SDF referred to a PGT-A (preimplantation genetic testing for aneuploidies) cycle.

Potential utility of a non-invasive menstrual blood immunophenotype analysis in reproductive medicine.

Purpose Can a comprehensive flow cytometry panel be used to assess immunophenotype profiles in menstrual blood of patients experiencing reproductive failure and age matched controls of proven fertility? Methods 58 recurrent pregnancy loss and repeated implantation failure patients, along with 15 age matched controls of proven fertility, had menstrual blood samples obtained within the first 24 hours of the onset of menstruation to non-invasively assess the local immunophenotype. Using a comprehensive multi-parameter flow panel the lymphocyte sub-populations were described and compared. Results Relative to well established peripheral blood immunophenotyping values, distinct lymphocyte population differences were noted between the subgroups. The ratios of CD4+ and CD8+ T-cells were inverted relative to peripheral blood and uterine NK cells represented by CD56bright were distinctly visualised, emphasising the distinction of menstrual and peripheral blood. Relative to controls there were marked increases in CD3+ve T-cells (p=0.009), CD4:CD8 ratio (p=0.004), CD19 B-cells (p=0.026) and CD56dim NK's (p=0.002) in the reproductive failure cases. Conclusions Flow cytometric evaluation can provide a rapid and objective analysis of lymphocyte subpopulations in many forms of tissue and fluid. The findings show significant variations in cellular composition of immune cells indicating a distinct compartment, with differences between cases and controls. Immunological assessment of the menstrual blood immunophenotype, in clinically appropriate patients, may provide insight into the aetiology of adverse reproductive outcome, without the risks and inconveniences associated with a more invasive endometrial biopsy.

EFFECTS OF AUTOLOGOUS PLATELET-RICH PLASMA IN WOMEN WITH POI AND POR

As the age of women undergoing IVF increases, the most common cause of repeated implantation failure has become ovarian dysfunction. A breakthrough treatment is needed to restore decreased ovarian function, improve oocyte quality and quantity, or resume ovulation in premature ovarian insufficiency (POI). The purpose of this study was to determine the efficacy of intraovarian autologous platelet-rich plasma (PRP) on ovarian folliculogenesis in women with poor ovarian responder (POR) and POI

ASSOCIATION BETWEEN CYTOKINE LEVELS IN BLOOD PLASMA AND PBMC CULTURE MEDIA

Intrauterine administration of human chorionic gonadotropin (hCG) activated peripheral blood mononuclear cells (PBMC) has positive effect on embryo implantation in repeated implantation failure patients. This effect is mainly caused by the PBMC cytokine production. The aim of this study was to find association between the tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), interleukin 4 (IL4) and interleukin 10 (IL10) plasma levels and their respective concentrations in the media of cultivated PBMC alone or treated with hCG.

Correlation between Endometrial Vascular Endothelial Growth Factor Expression and Pregnancy Outcome of Frozen-Thawed Embryo Transfer in Patients with Repeated Implantation Failure.

Objective Vascular endothelial growth factor (VEGF) is a well-known angiogenic factor that is essential to numerous physiological and pathological processes. VEGF also contributes to embryo implantation by promoting embryo development, enhancing endometrial receptivity (ER), and promoting interactions between the endometrium and developing embryo. Changes in VEGF expression are linked to repeated implantation failure (RIF). Control endometrial tissues demonstrated an increase in VEGF expression during the implant window period, which promoted early villous vascularization and embryo implantation. The purpose of this study is to investigate the relationship between RIF and the expression of ER markers, such as VEGF during the implantation window stage. Methods The Yinchuan Maternal and Child Health Hospital collected 192 cases of FET endometrial tissues in the implantation window stage between January 2019 and December 2021. Immunohistochemistry was utilized to measure the levels of VEGF expression in patients with RIF (RIF group, n = 82) and patients with a successful pregnancy (control group, n = 110). The relationship between VEGF and the RIF group was analyzed using Spearman's correlation coefficient. Results VEGF levels were significantly lower during the implantation window stage (P < 0.05). Conclusion VEGF was expressed in planting window stage. The decrease of VEGF during the implantation window was correlated with RIF.

Network Pharmacology and Molecular Docking Approach to Reveal the Immunotherapeutic Mechanism of Cuscutae Semen in Treating Thin Endometrium.

Objective Thin endometrium is considered as a leading cause of infertility, recurrent pregnancy loss, and repeated implantation failure. The seed of Cuscutae Semen (CS) has been used to prevent aging and improve sexual function in Traditional Chinese Medicine. However, the pharmacological mechanism of CS in preventing and treating thin endometrium remains to be elucidated. Methods Three public databases, TCMSP, GeneCards, and OMIM, were searched to collect the main active compounds and putative molecules of CS, as well as the targets of thin endometrium, respectively. The CS and thin endometrium common targets were subject to protein-protein interaction (PPI) analysis followed by functional enrichment analysis. The best binding mode of CS compounds and common target proteins was evaluated by molecular docking and analysis in the AutoDockTools. Results In total, 11 main active compounds, 102 drug target proteins, and 70 CS and thin endometrium common targets were identified. There were 68 nodes with 722 edges in the PPI network; HIF1A, MYC, ESR1, and EGFR were the top 4 targets. After functional enrichment analysis, it was revealed that the therapeutic effects of active compounds of CS on thin endometrium were achieved through cellular response to chemical stress, transcription regulator, DNA-binding transcription factor binding, chemical carcinogenesis-receptor activation, lipid, and atherosclerosis. The molecular docking analysis revealed that the 3 active compounds of CS, quercetin, matrine, and isorhamnetin, have good binding ability with their targets, HIF1A, MYC, ESR1, and EGFR. Conclusion Our study uncovers the main active compounds in CS and their corresponding targets related to thin endometrium which explains the pharmacological mechanism underlying therapeutic effects of CS on thin endometrium.

Preimplantation genetic testing for aneuploidy improves clinical outcomes in patients with repeated implantation failure

Objective:The objective of this study is to study whether preimplantation genetic testing for aneuploidy (PGT-A) improves the clinical outcomes of infertile patients with repeated implantation failure (RIF) undergoing frozen-thawed embryo transfer.Methods:This is a retrospective analysis of clinical pregnancy, live birth, miscarriage rates, and obstetric and perinatal outcomes of women with RIF with or without PGT-A. Statistical analyses of categorical data were performed using propensity score matching (PSM), χ2test, and Student’sttest.Results:We enrolled 466 patients with RIF, of which, 209 were in the RIF-PGT-A group. The rate of euploid blastocysts was significantly associated with age and day 5 or 6 blastocysts. There were significant differences between the RIF-PGT-A group and the RIF-non-PGT-A group across several parameters. After PSM, positive serum human chorionic gonadotropin (56.9% and 33.9%,P &lt;0.01), clinical pregnancy (49.5% and 31.2%,P &lt;0.01), live birth (43.1% and 25.7%,P &lt;0.01), and fetal heart rates (50.0% and 29.8%,P &lt;0.01) per transfer were significantly higher in the RIF-PGT-A group.Conclusion:Elective single-embryo transfer PGT-A can minimize the risk of obstetric and perinatal outcomes, especially fetal body weight, in women with RIF. Additionally, PGT-A can significantly improve pregnancy and live birth rates.

The Effect of Intrauterine Infusion of Plasma Enriched Platelet on Live Birth Rate in Patients with Implantation Failure: A Retrospective Uncontrolled Study

Background & Objective: Autologous platelet-rich plasma consists of concentrated autologous plasma and several cytokines and growth factors released by activated platelets in injury and inflammation. There is an increasing trend towards the effectiveness of intrauterine PRP infusion in repeated implantation failure patients. The aim of the present study was to describe the impact of intrauterine platelet-rich plasma infusion on the live birth rate in patients with repeated implantation failure.Materials & Methods: The present retrospective uncontrolled study was performed on 96 patients with more than two failed intracytoplasmic sperm injection cycles at Mehr medical institute between 2019 and 2021. Forty-eight hours before embryo transfer, patients received 1 mL lympho-platelet-rich plasma through an intrauterine insemination catheter. Patients were evaluated for pregnancy rate. Endometrial preparation for frozen-thawed embryo transfer was performed.Results: Participants' basal and stimulation characteristics, including gonadotropin dosage, the total number of oocytes, metaphase II oocytes and embryos, endometrial thickness, embryo transfer, quality of transferred embryos, and blastocyst transfer rate were evaluated. A total of 33 and 27 chemical (34.3%) and clinical pregnancies (28.1%) were achieved. Twenty (20.8%) and nineteen (20%) cycles resulted in ongoing pregnancies or live births, respectively. Conclusion: The current study suggests that platelet-rich plasma infusion 48 hours before frozen-thawed embryo transfer may be a good option for repeated implantation failure patients and results in 20% live birth.

OP-25 - Do abnormalities in an endometrial microbiota affect the pregnancy outcome of assisted reproductive technology in reproductive immunity?

Recently, the relationship between endometrial microbiota and repeated implantation failure (RIF) has been reported. However, it is still unclear the effect of the microbiota on pregnancy outcome of the patients with the assisted reproductive technology, especially a frozen embryo transfer (FET) treatment. We included 802 cycles with clinical results of pregnancy outcome after FET treatment at our clinic from December 2018 to January 2021. Endometrium fluid was collected before FET and microbiota was examined. We examined the relationship between endometrial microbiota and pregnancy outcome in 463 cycles in which the endometrial microbiome test results were available, pregnancy outcome was known, CD138 negative, and less than 38years (229 cycles were positive pregnancy and 234 cycles were negative pregnancy). Sampling was performed carefully avoiding contamination before FET treatment. Extracted genomic DNA was sequenced for the region of 16 S ribosomal RNA using next-generation sequencer (Endometrial microbiome test, Varinos, Japan). The sequencing data was assigned to bacterial taxonomy and the background-contaminated bacteria were excluded from the microbiome profile. Lactobacillus abundance was calculated. After concordance of patient background, Lactobacillus abundance between successful and unsuccessful pregnancy group was compared. We investigated the effect of microbiota in EF, especially Lactobacillus, for the infertile female with FET as an assisted reproductive treatment with large amount of cycles (802 cycles). First, all of cycles were divided into a successful pregnancy group (305/802) and an unsuccessful pregnant group (497/802), and then the abundance of Lactobacillus was analyzed (the pregnancy ratio was 38.0%). The mean abundance of Lactobacillus in EF was significantly higher in the successful pregnant group than in the unsuccessful pregnant group (70.2% ± 40.5 versus 63.5% ± 43.1, p=0.007). To further refine the conditions of each cycle, patient backgrounds of each cycle were compared. There was a significant difference in age and CD138 results between the successful pregnant group and unsuccessful pregnant group, so the analysis was performed with the limited cycles with CD138-negative and under 38 years (229/463 versus 234/463, respectively). However, even under this strict condition, the mean abundance of Lactobacillus was significantly higher in the pregnant group than in the unsuccessful pregnant group (72.1% ± 39.7 versus 61.1% ± 44.4, p=0.003). These results suggest that the endometrial environment with high Lactobacillus abundance is more conducive to pregnancy in FET treatment. The results of this study suggest that the endometrial microbiota at the time of embryo implantation, especially Lactobacillus genus, is highly relevant to pregnancy outcome, implying that disruption of the intrauterine microbiota may cause infertility. We need further studies to improve the endometrial microbiota of infertile women.

Is Endometrial Receptivity Assay (ERA) Useful in Patients with Repeated Implantation Failure Undergoing Single, Autologous Euploid Embryo Transfer?

Background: Our aim in this study was to evaluate whether endometrial receptivity assay (ERA) test improves single, autologous euploid frozen-thawed embryo transfer (FET) outcomes in patients with repeated implantation failure. Methods: This was a retrospective cohort study which was conducted in a University affiliated private hospital. The study included 135 patients with repeated implantation failure who underwent single, autologous euploid ERA adjusted and non-adjusted FET. Patients were stratified into three groups, patients with receptive endometrium based on the ERA test, patients with non-receptive endometrium based on the ERA test and patients who did not receive the ERA test (control group). The three groups were compared in terms of FET outcomes. Results: Of 135 patients, 73 had the ERA test results available and 62 did not have the ERA test. Of 73 patients, 28 had non-receptive endometrium and 45 had receptive endometrium. The three groups are all the same in terms of age, body mass index, type of infertility, duration of infertility, number of previously embryo transfers and infertility causes (p &gt; 0.05). Live birth rates were 46%, 50% and 51% for receptive, non-receptive and control groups, respectively (p &gt; 0.05). Implantation and clinical pregnancy rates were similar between the groups, as well. Conclusions: Adjusting the embryo transfer day according to the ERA test results seems to improve FET outcomes in patients with repeated implantation failure.

Genital tract dysbiosis in infertile women with a history of repeated implantation failure and pilot study for reproductive outcomes following oral enteric coating lactoferrin supplementation

We prospectively investigated if oral enteric coating lactoferrin supplementation improves the reproductive outcomes in infertile women with a history of repeated implantation failure (RIF) and non-Lactobacillus-dominant (Lactobacillus rate < 90%) microbiota (NLDM) in vaginal secretions (VS)/endometrial fluid (EF). Paired VS/EF samples were obtained from RIF women and control infertile women (non-RIF group) for microbiome analysis. Chronic endometritis (CE) was diagnosed histopathologically and hysteroscopically. In a pilot study, oral enteric coating lactoferrin (700mg/day, at least 28 consecutive days) was administered to eligible patients with NLDM in VS/EF. Their reproductive outcomes in the subsequent vitrified-warmed embryo transfer cycles were followed up. While CE was more prevalent (OR 2.41, 95% CI 1.02-5.63, p = 0.042) in the RIF group (29.1%, n = 117) than in the non-RIF group (14.5%, n = 55), The NLDM rate was similar between the two groups (44.4 vs 52.7%). Lactoferrin supplementation improved NLDM in 43.2% of RIF women (n = 37). Within the RIF group, the live birth rate in the subsequent cycles was higher (OR 10.67, 95% CI 1.03 - 110.0, p = 0.046) in women with improved microbiota (57.1%, n = 14) than in those with unimproved microbiota (11.1%, n = 9). Unlike CE, NLDM was not unique to RIF but was common in infertile women. Although the therapeutic effect of the oral lactoferrin supplementation on NLDM was limited in a pilot study, the reproductive outcomes were better in RIF women who overcame NLDM than in those who failed. Randomized controlled trials are required to confirm the results. TRIAL REGISTRATION NUMBER AND DATE FOR PROSPECTIVELY REGISTERED TRIALS: UMIN-CTR 000036990, June 7, 2019.

Role of endometrial microRNAs in repeated implantation failure (mini-review).

MicroRNAs (miRNAs) play various roles in the implantation and pregnancy process. Abnormal regulation of miRNAs leads to reproductive disorders such as repeated implantation failure (RIF). During the window of implantation, different miRNAs are released from the endometrium, which can potentially reflect the status of the endometrium for in vitro fertilization (IVF). The focus of this review is to determine whether endometrial miRNAs may be utilized as noninvasive biomarkers to predict the ability of endometrium to implant and provide live birth during IVF cycles. The levels of certain miRNAs in the endometrium have been linked to implantation potential and pregnancy outcomes in previous studies. Endometrial miRNAs could be employed as non-invasive biomarkers in the assisted reproductive technology (ART) cycle to determine the optimal time for implantation. Few human studies have evaluated the association between ART outcomes and endometrial miRNAs in RIF patients. This review may pave the way for more miRNA transcriptomic studies on human endometrium and introduce a specific miRNA profile as a multivariable prediction model for choosing the optimal time in the IVF cycle.

Lincomycin Administration against Persistent Multi-Drug Resistant Chronic Endometritis in Infertile Women with a History of Repeated Implantation Failure

Chronic endometritis (CE) is an infectious disease of the uterine lining, which is characterized by endometrial stromal plasmacyte (ESPC) infiltration. CE is often seen in infertile women with a history of repeated implantation failure (RIF) following an in vitro fertilization-embryo transfer program, recurrent pregnancy loss, and unknown etiology. Oral antibiotic agents, such as doxycycline, metronidazole, ciprofloxacin, azithromycin, and moxifloxacin, have been prescribed and are effective in the treatment of CE. Multi-drug resistance (MDR), however, is an emerging issue, as in other medical fields. We report six cases of persistent MDR-CE in infertile women who were resistant to all the aforementioned antibiotic agents. The bacterial genera and microbial communities unique to persistent MDR-CE were not identified in their vaginal secretions and/or endometrial fluid. Oral lincomycin administration (14 days, 1500 mg/day) was effective in the eradication of ESPCs in these women. In the embryo transfer cycles following histopathologic confirmation of cure (elimination of ESPCs) of persistent MDR-CE, three out of them had a successful live birth.

The outcomes of sequential embryo transfer in patients undergoing in vitro fertilization with frozen-thawed embryos: A retrospective study.

To explore whether sequential embryo transfer benefits patients with repeated implantation failure (RIF) undergoing frozen-thawed embryo transfer (FET) cycles. We included 311 patients with a history of RIF in this retrospective study. We did sequential transfers with a cleavage embryo on day 3 and a blastocyst on day 5 in 77 patients; blastocyst transfers with two blastocysts on day 5 in 80 patients; and cleavage embryo transfers with two cleavage embryos on day 3 in 154 patients. We compared clinical outcomes between the three groups. The clinical pregnancy rate was comparable between the blastocyst transfer group (48.8%), the sequential transfer group(48.1%) and the cleavage embryo transfer group (48.1%). There was no statistically significant difference found (p > 0.05). The ongoing pregnancy and multipregnancy rates were also comparable between the three groups (p > 0.05). The early miscarriage rate was significantly higher in the sequential transfer group (32.4%) compared with the blastocyst group (12.8%) and the cleavage embryo group (12.2%) (p < 0.05). However, after adjusting for confounders, there was no significant difference in early miscarriage rates in the sequential transfer group compared with the blastocyst group (odds ratio [OR], 2.97; 95% confidence interval [CI], 0.85-9.24; p=0.07) and the cleavage embryo group (OR, 3.03; 95% CI, 0.94-8.06; p=0.08). Sequential embryo transfer failed to improve clinical outcomes for patients with RIF.

P-410 Repeated unsuccessful embryo transfers and inflammatory parameters in serum before IVF start and in follicle fluid at OPU

Abstract Study question Is there a difference in cytokine Th1/Th2 ratio in women with repeated implantation failure (RIF) compared to women with a successful IVF treatment? Summary answer Proinflammatory activity in both serum and follicle fluid (FF) differs in RIF patients and patients with successful IVF treatment. What is known already Unexplained recurrent implantation failure (RIF) is generally used to describe patients who have not become pregnant after repeated embryo transfers (ETs) of good quality embryos (GQE) during IVF treatment. There is no unison definition. Implantation requires a receptive endometrium. Receptivity is modulated by cytokines amongst other factors. Cytokines are signaling molecules mainly produced by T-helper lymphocytes (Th; CD3+/CD4+) and macrophages. The guiding of the blastocyst to the endometrial lining, the trophoblast cell invasion and the subsequent remodeling of the damaged endometrial tissue all require proinflammatory cytokines (Th1 dominant), whereas the sustainment of pregnancy requires an anti-inflammatory state (Th2 dominant). Study design, size, duration Retrospective study based on material collected during a larger study including 384 women enrolled during 2007-2016. Inclusion criteria were: &amp;lt;40 years of age, body mass index &amp;lt;30kg/m2, non-smoking, regular menstruation cycle of 21-35 days and bilateral ovaries. Serum was obtained before treatment and FF was collected during OPU in the first IVF cycle. Participants/materials, setting, methods Patients with no implantation after ≥3 ETs with fresh or frozen GQEs and any indication were included in the RIF group (n = 29). Women with achieved pregnancy after ≤3 ETs, with an indication of male factor, were selected as controls (n = 26). Cytokines were analysed with the MSD Proinflammatory Human Kit 1 (MSD, USA; IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF-α). Th1/Th2 ratios were calculated and compared with the Mann-Whitney U test. Main results and the role of chance In serum there was a difference between RIF and non RIF in ratios for IFN-γ/IL10 (p = 0,01), IL-1β/IL10 (p = 0,04), IL2/IL10 (p = 0,00), TNF-α/IL10(p = 0,02), IFNγ/IL13(p = 0,01), IL12/IL13 (p = 0,02), IL2/IL13 (p = 0,00) and TNF-α/IL13 (p = 0,00) (p &amp;lt; 0,05 for all), where the control group all had higher ratios, eg. a shift towards a Th1 profile before treatment start. In FF there were differences in ratios between IL2/IL10 (p = 0,02), IL-8/IL-10 (0,02), TNF-α/IL10 (p = 0,02), IFNγ/IL13 (p = 0,01) and TNF-α/IL13 (p = 0,03). The RIF group had higher ratios of all these except for IFNγ/IL13, indicating that they had a shift towards a Th1 profile in their FF. The RIF patients had a median age of 33 years, whilst the median age in the controls was 30 years (p = 0.01), there was no difference in BMI or cycle length. Using inclusion and exclusion criteria regarding lifestyle factors and a strict definition of RIF and controls brings strength to the results and minimizes the role of chance. Also, there were no participants with chronic inflammatory disease. Limitations, reasons for caution The FF was pooled so the cytokine concentrations in the follicle containing the oocyte used for fertilization and ET was not determined. The age difference between the groups could have an effect. Yet, the median was below 35 years, where a decline in implantation and pregnancy rates are found. Wider implications of the findings Women at risk of immune related RIF could be identified proactively. Because it is taken at a timepoint closer to implantation, ratios in FF are specifically interesting as risk markers. Women with RIF and a Th1 shift in FF could be offered immune modulating therapy within a research setting. Trial registration number not applicable

P-380 Single-cell transcriptome analysis reveales that expression changes of the endometrium in repeated implantation failure are altered by HPV-mediated CXCL chemokine secretion

Abstract Study question What are the mechanisms and molecular expression patterns of reduced endometrial receptivity in repeated implantation failure (RIF) after human papillomavirus (HPV) infection? Summary answer The single-cell transcriptomic analysis identifies the expression changes of endometrium in RIF via HPV-mediated CXCL chemokines secretion in single-cell resolution. What is known already Regardless of the advance of in vitro fertilization (IVF), RIF is still a formidable challenge for couples and physicians in clinical treatment. In infertile couples, a reduction in natural and assisted cumulative pregnancy rate and an increase in miscarriage rate are related to the HPV infection. Study design, size, duration Cross-sectional clinical studies with 322 infertile couples undergoing IVF were integrated to demonstrate the associations between HPV infection and reproductive outcomes (pregnancy rate and miscarriage). Descriptive analysis of single-cell transcriptome data of uteruses, and transcriptome profiles of mid-secretory endometrium from 16 healthy fertile women and 38 repeated IVF failure women were analyzed to identify the expression patterns of endometrium in RIF. In vitro assays were used to validate the expression patterns in endometrium. Participants/materials, setting, methods 322 infertile couples, single-cell transcriptome data of uteruses (human and mouse), and transcriptome profiles of endometrium (16 normal vs. 38 RIF) were used to analyze the association between HPV infection and reduced endometrial receptivity. HPV genes (E1, E2, E4, and E5) were transfected into a human normal endometrial epithelial cell line (hEM3), and immunohistochemistry, Westerns, quantitative PCR were used to validate the changes of CXCL chemokines in the endometrium in vitro. Main results and the role of chance Integrated cross-sectional studies demonstrate that HPV+ women exhibit a decreased pregnancy rate (83.09%) as compared with HPV- women (55.17%, P &amp;lt;0.001), and a higher miscarriage rate (62.5% vs. 16.7%, P &amp;lt;0.001) and the relative risk of spontaneous abortion (odd ratio=2.84, P &amp;lt;0.0001) were observed in HPV+ women. Transcriptome profiling analysis identified the enrichment of the processes related to viral protein interaction with cytokine and cytokine receptor and cytokine-cytokine receptor interaction, especially in the CXCL chemokine family. Further analysis of single-cell transcriptome demonstrated that the changed expression patterns were associated with endometrial epithelial cells and immune cells, including macrophage dendritic cells, monocytes, and granulocytes. Moreover, in vitro assays validated the HPV-mediated CXCL chemokines secretion, which played the role in recruiting immune cells. Limitations, reasons for caution The current findings are based on the single-cell profiling analysis in normal endometrium. In addition, the in vivo response of the HPV infection may differ from the in vitro assay, which should be validated in the HPV infection couples. Wider implications of the findings Our study demonstrated the expression changes of endometrium in RIF via HPV-mediated CXCL chemokines secretion, which provided insight into the mechanisms of HPV-induced reduced endometrial receptivity in single-cell resolution. Trial registration number not applicable

P-327 The impact of chronic endometritis disease on endometrial receptivity gene expression in women with repeated implantation failure

Abstract Study question Does chronic endometritis (CE) have an impact on the expression of the genes involved in the embryo implantation? Summary answer The mRNA expression of genes involved in embryo implantation (HOXA10, HOXA11, BTEB1 and LIF) does not change in patients with and without CE. What is known already CE is an inflammatory disorder of the endometrium with a detrimental effect on embryo implantation and its prevalence in women with repeated implantation failure (RIF) is up to 30%. During the implantation window, the expression of some genes plays a key role in determining the success of embryo implantation. The homeobox genes HOXA10, HOXA11 and the basic transcriptional element binding protein 1 (BTEB1) gene are essential for endometrial cells growth regulation and for embryonic development and the leukaemia inhibitory factor (LIF) has a role in the embryo–endometrium interaction. Study design, size, duration A single centre-prospective case-control study was conducted at the Women’s and Children’s Health Department at Padua University, on infertile women with RIF from June 2020 to November 2021. 29 women with RIF were prospectively enrolled. Participants/materials, setting, methods An endometrial biopsy using a Novak curette was performed for each patient and the obtained tissue samples were divided in two aliquots, one for immunohistochemistry for histological examination (endometrial dating and CE diagnosis) and one for RNA extraction and gene expression analysis that has been performed using RT-PCR. Sample of patients with and without CE were compared using non-parametric Mann–Whitney U-test. A p-value &amp;lt;.05 was considered as statistically significant. Main results and the role of chance Enrolled women were divided in two groups according to the histological diagnosis of CE: 13 patients with CE, 16 without CE. In all samples, appropriate histological dating (WOI) was evaluated according to Noyes criteria. After the comparison of all investigated genes (HOXA10, HOXA11, BTEB1 and LIF), no significant difference in mRNA expression was detected between women with and without CE (p value &amp;gt;.05). In the literature, defective endometrial expression of HOXA10, HOXA11 and LIF genes has been associated with abnormal implantation and the reduced expression of BTEB1 gene results in subfertility and progesterone resistance. Although the negative role of CE in altering embryo implantation is known, our results suggest that the CE has no effect on the expression of HOXA10, HOXA11, BTEB1 and LIF genes. Probably the detrimental effect of CE on embryo implantation does not act through HOXA10, HOXA11, BTEB1 and LIF gene expression. Limitations, reasons for caution This is a non-randomized observational study with a limited number of patients. Further studies are needed to confirm our data with immunohistochemistry evaluation to define the protein expression levels of the investigated genes. Wider implications of the findings Understanding the pathogenic mechanism of CE on endometrial receptivity is crucial for identifying markers that best correlate with possible implantation failure and for identifying the appropriate therapy to treat the disease and restore the embryo implantation capacity. Trial registration number not applicable

P-383 Endometrial scratching: A strategy to stimulate the angiogenesis aspect of implantation in unexplained, repeated implantation failure

Abstract Study question Does endometrial scratching serve as an angiogenic stimulator during implantation in unexplained, repeated implantation failure (uRIF) patients? Summary answer Endometrial scratching can reimburse and regulate genes involved in both endometrial angiogenesis and receptivity. What is known already The vascular endothelial growth factor (VEGF), as an angiogenic factor, is well-known for its function in a variety of physiological and pathological aspects of female reproduction. Following the embryo transfer in the In-Vitro Fertilization cycles, the most challenging clinical obstacle to overcome is RIF. Endometrial scratching is a technique considered as an option for enhancing the embryo implantation of uRIF patients by modulating the different cellular and molecular components regulating endometrial receptivity before and during implantation. Study design, size, duration Twenty uRIF patients were enrolled based on a randomized controlled trial (RCT) to study the expression of genes involved in endometrial angiogenesis and receptivity functions (VEGFR1, VEGFR2, COL18A1, E-cadherin, FGF1) following ES. Ten uRIF patients were randomly assigned to the intervention group (twice endometrial sampling in the follicular and luteal phases) and ten in the control group (only luteal phase sampling) prior to the ovarian stimulation cycle. Participants/materials, setting, methods Women who failed to conceive following three or more IVF/ICSI cycles, high-quality embryo transfer, and at least one blastocyst embryo transfer cycle met the inclusion criteria. Post-biopsy endometrial samples were divided evenly. One component was placed in 10% formalin for histology dating and another in RNALater for genomic analysis and preserved at -80 °C for RNA extraction. Main results and the role of chance The expression levels of VEGFR1 and VEGFR2 (both as the receptors of VEGF) were significantly increased after ES (P &amp;lt;0.05). The mRNA expression level of COL18A1 was significantly decreased after ES. COL18A1 is an inhibitor of VEGF which can impede VEGF-mediated signaling and endothelial cell proliferation, via blocking of its receptor specifically VEGFR2. The E-cadherin expression level was significantly decreased (P &amp;lt;0.05) in the endometrium of the intervention group after scratching. E-cadherin is a cell-cell adhesion molecule that has a pivotal role in maintaining normal epithelial architecture, establishing cell polarity, glandular differentiation, and morphogenesis. Data showed that Fibroblast Growth Factor1 (FGF1) expression level was significantly increased in the intervention group (P &amp;lt;0.05). FGF1 promotes the formation of blood vessels which improves endometrial trophoblastic interaction and affects embryo implantation. Limitations, reasons for caution To further understand the function of these changes, it is preferable to compare the data with endometrium from a fertile group, which is ongoing. Wider implications of the findings On the timeline of embryo implantation, angiogenic factors (e.g., VEGFR1 and VEGFR2), their inhibitors (e.g., E-cadherin and COL18A1), and activators (e.g., FGF1) work together to establish a dynamic environment responsible for endometrial receptivity, which has been compromised in the endometrium of RIF patients.ES can reimburse and regulate these angiogenesis processes. Trial registration number IRCT20210316050723N1

O-256 Effect of an endometrial microbiota on pregnancy outcome of frozen embryo transfer (FET) cycles

Abstract Study question Is specific microbiota in the endometrial fluid (EF) associated with pregnancy outcome in a frozen embryo transfer (FET) cycles? Summary answer Lactobacillus had high abundance in the microbiota of endometrial fluid of patients with successful pregnancy after FET treatment. What is known already Recently, the relationship between endometrial microbiota and repeated implantation failure (RIF) has been reported. While Lactobacillus-dominated microbiota (LDM, defined as &amp;gt; = 90 % Lactobacillus species) in the EF of the receptive phase was reported to be associated with favorable reproductive outcome, non-LDM (&amp;lt; 90 % Lactobacillus species) was found to decrease implantation, clinical pregnancy, ongoing pregnancy, and live birth rates. However, it is still unclear the effect of the microbiota on pregnancy outcome of the patients with the assisted reproductive technology, especially a frozen embryo transfer (FET) treatment. Study design, size, duration We included 802 cycles with clinical results of pregnancy outcome after FET treatment at our clinic from December 2018 to January 2021. Endometrium fluid was collected before FET and microbiota was examined. We examined the relationship between endometrial microbiota and pregnancy outcome in 463 cycles in which the endometrial microbiome test results were available, pregnancy outcome was known, CD138 negative, and less than 38years (229 cycles were positive pregnancy and 234 cycles were negative pregnancy). Participants/materials, setting, methods Sampling was performed carefully avoiding contamination before FET treatment. Extracted genomic DNA was sequenced for the region of 16S ribosomal RNA using next-generation sequencer (Endometrial microbiome test, Varinos, Japan). The sequencing data was assigned to bacterial taxonomy and the background-contaminated bacteria were excluded from the microbiome profile. Lactobacillus abundance was calculated. After concordance of patient background, Lactobacillus abundance between successful and unsuccessful pregnancy group was compared. Main results and the role of chance We investigated the effect of microbiota in EF, especially Lactobacillus, for the infertile female with frozen embryo transfer (FET) as an assisted reproductive treatment with large amount of cycles (802 cycles). First, all of cycles were divided into a successful pregnancy group (305/802) and an unsuccessful pregnant group (497/802), and then the abundance of Lactobacillus was analyzed (the pregnancy ratio was 38.0%). The mean abundance of Lactobacillus in EF was significantly higher in the successful pregnant group than in the unsuccessful pregnant group (70.2% 40.5 versus 63.5% 43.1, p = 0.007). To further refine the conditions of each cycle, patient backgrounds of each cycle were compared. There was a significant difference in age and CD138 results between the successful pregnant group and unsuccessful pregnant group, so the analysis was performed with the limited cycles with CD138-negative and under 38 years (229/463 versus 234/463, respectively). However, even under this strict condition, the mean abundance of Lactobacillus was significantly higher in the pregnant group than in the unsuccessful pregnant group (72.1% 39.7 versus 61.1% 44.4, p = 0.003). These results suggest that the endometrial environment with high Lactobacillus abundance is more conducive to pregnancy in FET treatment. Limitations, reasons for caution The limitation of this study is that the design is not a randomized controlled one, although it is prospective. Wider implications of the findings The results of this study suggest that the endometrial microbiota at the time of embryo implantation, especially Lactobacillus genus, is highly relevant to pregnancy outcome, implying that disruption of the intrauterine microbiota may cause infertility. We need further studies to improve the endometrial microbiota of infertile women. Trial registration number not applicable