Assessment of immune cells in the uterine fluid at the time of the embryo transfer.

PROBLEM: Although endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium that affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation. METHOD OF STUDY: This study was approved by the Institutional Review Board (# 225589). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic-based fertility center. All patients underwent at least one In Vitro Fertilization (IVF) cycle and have frozen embryos. Twenty-four participants were recruited for this study which was conducted during the frozen embryo transfer (FET) cycle regardless of the outcome of previous cycles. Two samples were acquired from each subject, denoted as lower and upper. A trial transfer catheter was introduced under ultrasound guidance into the lower uterine segment. Upon removal, the tip was rinsed in IMDM medium containing 10% FBS (lower uterus). A transfer catheter was then loaded with the embryo that was placed in the upper uterus under ultrasound guidance. The tip of the transfer catheter was rinsed in separate aliquot of the above media (upper uterus). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56 and acquired on Sony SP6800 Spectral Analyzer. RESULTS: Upon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.125 × 106 cells ± SD 0.32), (0.123 × 106 cells ± SD 0.12), respectively. Among total viable cells, there was no significant difference in both percent and number of CD45+ cells between the upper and lower uterus (9.88% ± 6.98 SD, 13.67% ± 9.79 SD, p = .198) respectively. However, there was significantly higher expression of CD3+ (p = .006), CD19+ (p = .032) and CD14+ (p = .019) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p = .009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells (CD4+CD25+CD127-), NK cells (CD56+), neutrophils (CD66b+) and FcγRIII+ cells (CD16+) between upper and lower uterus. CONCLUSIONS: We believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cells will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes.

Correlation of peripheral blood and endometrial immunophenotyping in ART: is peripheral blood sampling useful?

PURPOSE: Using a comprehensive flow cytometric panel, simultaneously obtained mid-luteal immunophenotypes from peripheral blood and endometrium were compared and values correlated. Is a peripheral blood evaluation of reproductive immunophenotype status meritorious relative to local endometrial evaluation to directly assess the peri-implantation environment? METHODS: Fifty-five patients had a mid-luteal biopsy to assess the local endometrial immunophenotype, while simultaneously providing a peripheral blood sample for analysis. Both samples were immediately assessed using a comprehensive multi-parameter panel, and lymphocyte subpopulations were described and compared. RESULTS: Distinct lymphocyte proportions and percentage differences were noted across the two compartments, confirming the hypothesis that they are distinct environments. The ratio of CD4 + to CD8 + T cells were reversed between the two compartments, as were Th1 and Th2-type CD4 + T cell ratios. Despite these differences, some direct relationships were noted. Positive Pearson correlations were found between the levels of CD57 + expressing natural killer cells, CD3 + NK-T cells and CD4 + Th1 cells in both compartments. CONCLUSIONS: Flow cytometric evaluation provides a rapid and objective analysis of lymphocyte subpopulations. Endometrial biopsies have become the gold standard technique to assess the uterine immunophenotype in adverse reproductive outcome, but there may still a place for peripheral blood evaluation in this context. The findings demonstrate significant variations in cellular proportions across the two regions, but some positive correlations are present. Immunological assessment of these specific peripheral blood lymphocyte subtypes may provide insight into patients with potential alterations of the uterine immune environment, without the risks and inconveniences associated with an invasive procedure.

Transcriptome study of receptive endometrium in overweight and obese women shows important expression differences in immune response and inflammatory pathways in women who do not conceive.

Obesity and being overweight are growing worldwide health problems that also affect women of reproductive age. They impair women's fertility and are associated with lower IVF success rates. The mechanism by which increased body weight disrupts fertility has not yet been established. One possibility is that it affects the process of embryo implantation on the endometrial level. The purpose of our study was to determine the differences in enriched biological pathways in the endometrium of overweight and obese women undergoing IVF procedures. For this purpose, 14 patients (5 pregnant, 9 non-pregnant) were included in the study. Endometrial samples were obtained during the window of implantation and RNA sequencing was performed. There were no differences in general patient's and IVF cycle characteristics between pregnant and non-pregnant women. In the endometrial samples of women who did not conceive, pathways related to the immune response, inflammation, and reactive oxygen species production were over-expressed. Our findings show that the reason for implantation failure in overweight and obese women could lie in the excessive immune and inflammatory response at the endometrial level.

Human leukocyte antigen-C and killer immunoglobulin-like receptors in reproductive medicine. / Antígeno leucocitario humano C y receptores tipo inmunoglobulina en medicina reproductiva.

Proper communication between natural killer cells and the human leukocyte antigens of the embryonic trophoblast at the maternal-fetal interface during pregnancy is essential for successful reproduction. However, specific combinations of embryonic human leukocyte antigen-C with killer immunoglobulin-like receptors on decidual natural killer cells (the immunological code of pregnancy) can be associated with obstetric morbidity and pregnancy loss. This article presents an updated review of the mechanisms underlying the interaction between embryonic human leukocyte antigen-C and maternal killer immunoglobulin-like receptors and their relevance to the physiology and pathophysiology of human reproduction.

Una adecuada comunicación entre las células asesinas naturales en la interfase materno-fetal con las moléculas de los antígenos de histocompatibilidad del trofoblasto embrionario es clave en el éxito de la reproducción. Sin embargo, combinaciones de determinados antígenos leucocitarios humanos tipo C embrionarios con los receptores tipo inmunoglobulina presentes en las células asesinas naturales deciduales (el código inmunológico del embarazo), pueden asociarse con morbilidad obstétrica y pérdidas gestacionales. En este artículo se presenta una revisión actualizada de los mecanismos subyacentes a la interacción entre el antígeno de histocompatibilidad tipo C embrionario y los receptores tipo inmunoglobulina maternos, y su relevancia tanto en la fisiología como en la fisiopatología de la reproducción humana.

The Effects of Sperm and Seminal Fluid of Immunized Male Mice on In Vitro Fertilization and Surrogate Mother-Embryo Interaction.

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination's effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C-C, Im-C, C-Im, and Im-Im. The pre-implantation losses were significantly lower in the Im-C group than in the C-Im group. At the same time, the resorption rates reduced markedly in the C-Im compared to the Im-C group. Embryo and placenta weights were significantly higher in the Im-Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im-Im group, they were strongly correlated with embryo mass. The number-size trade-off was only significant in the Im-Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males' antigenic challenge on the IVF success rate.

Seminal Plasma and Seminal Plasma Exosomes of Aged Male Mice Affect Early Embryo Implantation via Immunomodulation.

Seminal plasma (SP), particularly SP exosomes (sExos), alters with age and can affect female mouse uterine immune microenvironment. However, the relationship between fertility decline in reproductively older males, and SP and sExos age-related changes, which may compromise the uterine immune microenvironment, remains unclear. The present study demonstrated that the implantation rate of female mice treated with SP from reproductively older male mice (aged-SP group) was lower than that of those treated with SP from younger male mice (young-SP group). RNA-sequencing analysis revealed altered levels of dendritic cell (DC)-related cytokines and chemokines in the uteri of the former group compared with those of the latter group. In vivo and in vitro experiments demonstrated a weaker inhibitory effect of aged SP on DC maturation than of young SP upon stimulation. After isolating and characterizing sExos from young and advanced-age male mice, we discovered that insemination of a subset of the aged-SP group with sExos from young male mice partially recovered the implantation rate decline. Additional in vivo and in vitro experiments revealed that sExos extracted from age male mice exerted a similar effect on DC maturation as SP of aged mice, indicating an age-related sExos inhibitory effect. In conclusion, our study demonstrated that age-related alterations of sExos may be partially responsible for lower implantation rates in the aged-SP group compared with those in the young-SP group, which were mediated by uterine immunomodulation. These findings provide new insights for clinical seminal adjuvant therapy.

CD19 and intraglandular CD163-positivity as prognostic indicators of pregnancy outcome in CD138-negative women with a previous fresh-embryo-transfer failure.

Many factors impede embryonic implantation, and excluding obvious known factors such as chronic endometritis, the immune status of the endometrium may be related to pregnancy. Although an abundantly large number of immune cells infiltrate the endometrium during the secretory phase, whether these immune cells can be used as a predictor of prognosis in ART has not yet been clarified. In the present study we therefore retrospectively analyzed 97 CD138-negative women with a previous fresh-embryo-transfer failure. We assessed the expression of CD56+ uNK cells, CD16+ NK cells, CD57+ NK cells, CD68+ pan-macrophages, CD163+ M2 macrophages, CD4+T cells, CD8+T cells, FOXP3+ regulatory T cells, and CD19+ B cells in the endometrium by IHC to evaluate mid-luteal endometrial immune cells as prognostic indicators of pregnancy outcome in the next frozen-embryo-transfer cycle. CD19-positive cells and the intraglandular CD163-positivity rate increased significantly in the clinically non-pregnant group (0.47 % vs. 0.20 %, P = 0.021; 61 % vs. 30 %, P = 0.017). The ratios of CD4/CD8 were also higher in the non-pregnant group (1.96 vs. 1.45, P = 0.005).The area under the ROC curve of CD19 cell number alone, the intraglandular CD163-positivity alone, and CD19 number combined with the intraglandular CD163-positivity were 0.692 (95 % CI, 0.55-0.834), 0.661 (95 % CI, 0.514-0.809), and 0.748 (95 % CI, 0.614-0.882), respectively. The optimal cut-off value of CD19 was 0.464 %, and the clinical pregnancy rate and live-birth rate diminished significantly when the CD19 level was above this cut-off value. Our study suggests that CD19-positive cells and intraglandular CD163-positivity can be used as prognostic indicators of pregnancy outcome in CD138-negative patients who experienced first-fresh-embryo transfer failure.

The effect of bta-miR-26b in intrauterine extracellular vesicles on maternal immune system during the implantation period.

Extracellular vesicles (EVs) in utero play a role in cellular interactions between endometrium-conceptuses (embryo plus extraembryonic membranes) during peri-implantation periods. However, how intrauterine EVs function on endometrium have not been well characterized. In our previous study, bta-miR-98 found in intrauterine EVs from uterine flushing fluids (UFs) on pregnant day 20 (a half day after initial conceptus attachment, P20) could regulate the maternal immune system and collaborate with other miRNAs and/or components of EVs for conceptus implantation. We, therefore, hypothesized that in addition to bta-miR-98, other miRNAs present in bovine intrauterine EVs may regulate the maternal immune system in the endometrial epithelium. A global analysis of differentially expressed proteins between EVs from P17 and P20 UFs revealed that components of intrauterine P20 EVs had the effect on the down-regulation of "neutrophil activation involved in immune response" and "neutrophil mediated immunity". In silico analyses predicted bta-miR-26b as one of potential miRNA to regulate maternal immune system. In our cell culture experiments, bta-miR-26b negatively regulated several immune system-related genes PSMC6, CD40, and IER3 in bovine endometrial epithelial cells. Our findings revealed that intrauterine EV-derived bta-miR-26b contributes to the down-regulation of the maternal immune system, allowing conceptus implantation to the uterine endometrium. Furthermore, our results suggest that intrauterine EVs extracted from P20 UFs could regulate neutrophils, the first line of immunological defense, to modulate endometrial immune and inflammatory responses for implanting conceptuses.

Integrated Insight into the Molecular Mechanisms of Spontaneous Abortion during Early Pregnancy in Pigs.

Due to the high rate of spontaneous abortion (SAB) in porcine pregnancy, there is a major interest and concern on commercial pig farming worldwide. Whereas the perturbed immune response at the maternal-fetal interface is an important mechanism associated with the spontaneous embryo loss in the early stages of implantation in porcine, data on the specific regulatory mechanism of the SAB at the end stage of the implantation remains scant. Therefore, we used high-throughput sequencing and bioinformatics tools to analyze the healthy and arresting endometrium on day 28 of pregnancy. We identified 639 differentially expressed lncRNAs (DELs) and 2357 differentially expressed genes (DEGs) at the end stage of implantation, and qRT-PCR was used to verify the sequencing data. Gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and immunohistochemistry analysis demonstrated weaker immune response activities in the arresting endometrium compared to the healthy one. Using the lasso regression analysis, we screened the DELs and constructed an immunological competitive endogenous RNA (ceRNA) network related to SAB, including 4 lncRNAs, 11 miRNAs, and 13 genes. In addition, Blast analysis showed the applicability of the constructed ceRNA network in different species, and subsequently determined HOXA-AS2 in pigs. Our study, for the first time, demonstrated that the SAB events at the end stages of implantation is associated with the regulation of immunobiological processes, and a specific molecular regulatory network was obtained. These novel findings may provide new insight into the possibility of increasing the litter size of sows, making pig breeding better and thus improving the efficiency of animal husbandry production.

Mapping themes trends and knowledge structure of trophoblastic invasion, a bibliometric analysis from 2012-2021.

OBJECTIVES: Trophoblastic invasion at the maternal-fetal interface can affect pregnancy outcomes. To describe an intuitive theme trends and knowledge structure of trophoblastic invasion-related literature from a bibliometric perspective, and provide researchers with new research hotspots. STUDY DESIGN: The literature form PubMed database related to trophoblastic invasion from January 1, 2012 to April 30, 2021 were extracted, and then biclustering analysis, co-word analysis, strategy diagram and social network analysis were performed to provide immature, or newly emerging research hotspots for researchers. RESULTS: A total of 96 high-frequency medical subjects heading terms were extracted and classified into 6 clusters. Themes in the first and second quadrant of strategy diagram, including trophoblasts metabolism, placenta metabolism, pre-eclampsia, etc., as the mature parts of the research on trophoblastic invasion have been well developed. On the other hand, themes in the third and fourth quadrants of strategy diagram, such as embryo implantation and trophoblasts immunology, pregnancy complication, matrix metalloproteinase and trophoblasts metabolism, habitual abortion and trophoblasts metabolism, etc., are immature themes. Social network analysis suggests that themes at the edge, such as habitual abortion / metabolism, placenta / immunology, natural killer cells / physiology, natural killer cells / immunology, embryo implantation / immunology, are considered new research hotspots and have considerable research space. CONCLUSION: By analyzing the research hotspots related to trophoblastic invasion, immature themes and emerging hotspots deserve more attention and can be considered as hints when launching new research projects.

Exploration of the potential roles of m6A regulators in the uterus in pregnancy and infertility.

Infertility is a prevalent female reproductive disease worldwide. Currently, there are many unknown etiologies of infertility. N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic mRNA. This study intended to investigate the implications of m6A regulators in the uterus for pregnancy and infertility. Pregnant ICR mice on days (D) 0, 4, 6, 10, and 15 were used to monitor m6A methylation in the uterus by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then m6A methylation regulators were detected by real-time quantitative PCR (qPCR), western blot and immunohistochemistry (IHC). We found that m6A levels increased and that m6A regulators were expressed differently in the uterus during pregnancy. Then, we acquired expression data from endometrial tissue from women with infertility and recurrent pregnancy loss from the Gene Expression Omnibus (GEO) database. The expression of m6A regulators in infertility was significantly dysregulated according to the data mining technique. Specifically, the mRNA levels of METTL16 (p = 0.0147) and WTAP (p = 0.028) were lower and those of ALKBH5 (p = 0.0432) and IGF2BP2 (p = 0.0016) were higher in the endometrium of infertile patients. Meanwhile, many immunity-related pathways are abnormal in infertility, such as cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity and leukocyte transendothelial migration. In conclusion, we found that the m6A levels in the uterus increased as pregnancy progressed, and these regulators were dysregulated in the endometrium of infertility patients. These results suggest that m6A methylation may be very important in the establishment of implantation and maintenance of pregnancy and may become a new direction for research on infertility.

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation.

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin ß3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

Sperm modulate uterine immune parameters relevant to embryo implantation and reproductive success in mice.

Seminal fluid factors modulate the female immune response at conception to facilitate embryo implantation and reproductive success. Whether sperm affect this response has not been clear. We evaluated global gene expression by microarray in the mouse uterus after mating with intact or vasectomized males. Intact males induced greater changes in gene transcription, prominently affecting pro-inflammatory cytokine and immune regulatory genes, with TLR4 signaling identified as a top-ranked upstream driver. Recruitment of neutrophils and expansion of peripheral regulatory T cells were elevated by seminal fluid of intact males. In vitro, epididymal sperm induced IL6, CXCL2, and CSF3 in uterine epithelial cells of wild-type, but not Tlr4 null females. Collectively these experiments show that sperm assist in promoting female immune tolerance by eliciting uterine cytokine expression through TLR4-dependent signaling. The findings indicate a biological role for sperm beyond oocyte fertilization, in modulating immune mechanisms involved in female control of reproductive investment.

Effect of blastocyst morphology and developmental speed on transfer strategy for grade "C" blastocyst in vitrified-warmed cycles.

BACKGROUND: High-quality single blastocyst transfer (SBT) is increasingly recommended to patients because of its acceptable pregnancy outcomes and significantly reduced multiple pregnancy rate compared to double blastocyst transfer (DBT). However, there is no consensus on whether this transfer strategy is also suitable for poor-quality blastocysts. Moreover, the effect of the development speed of poor-quality blastocysts on pregnancy outcomes has been controversial. Therefore, this study aimed to explore the effects of blastocyst development speed and morphology on pregnancy and neonatal outcomes during the frozen embryo transfer (FET) cycle of poor-quality blastocysts and to ultimately provide references for clinical transfer strategies. METHODS: A total of 2,038 FET cycles of poor-quality blastocysts from patients 40 years old or less were included from January 2014 to December 2019 and divided based on the blastocyst development speed and number of embryos transferred: the D5-SBT (n = 476), D5-DBT (n = 365), D6-SBT (n = 730), and D6-DBT (n = 467) groups. The SBT group was further divided based on embryo morphology: D5-AC/BC (n = 407), D5-CA/CB (n = 69), D6-AC/BC (n = 580), and D6-CA /CB (n = 150). RESULTS: When blastocysts reach the same development speed, the live birth and multiple pregnancy rates of DBT were significantly higher than those of SBT. Moreover, there was no statistical difference in the rates of early miscarriage and live birth between the AC/BC and CA/CB groups. When patients in the SBT group were stratified by blastocyst development speed, the rates of clinical pregnancy (42.44 % vs. 20.82 %) and live birth (32.35 % vs. 14.25 %) of D5-SBT group were significantly higher than those of D6-SBT group. Furthermore, for blastocysts in the same morphology group (AC/BC or CA/CA group), the rates of clinical pregnancy and live birth in the D5 group were also significantly higher than those of D6 group. CONCLUSIONS: For poor-quality D5 blastocysts, SBT can be recommended to patients because of acceptable pregnancy outcomes and significantly reduced multiple pregnancy rate compared with DBT. For poor-quality D6, the DBT strategy is recommended to patients to improve pregnancy outcomes. When blastocysts reach the same development speed, the transfer strategy of selecting blastocyst with inner cell mass "C" or blastocyst with trophectoderm "C" does not affect the pregnancy and neonatal outcomes.

JAK3 and PI3K mediates the suppressive effects of interferon tau on neutrophil extracellular traps formation during peri-implantation period.

Interferon tau (IFNτ) is the main maternal signal for pregnancy in ruminants and modulates the functions of various immune cells, including neutrophils. Neutrophil extracellular traps (NETs) are one of the main defence mechanisms of neutrophils. In this study, we observed higher (p < 0.01) ex-vivo NETs extrusion by blood neutrophils from day 16-18 post artificial insemination (AI) in non-inseminated and inseminated non-pregnant cows compared to pregnant cows. In vitro study also showed that IFNτ hampers NETs formation in dose and time dependent manner. The lowest (p < 0.01) NETs formation and the highest (p < 0.01) mRNA expression (RT-PCR) of IFNτ stimulated genes (ISG15, OAS1, MX1) were observed when neutrophil incubated with 9 ng/mL IFNτ for 3.5 h. Signalling cascades mediating IFNτ impairment of NETs formation were identified using inhibitors of JAK2, JAK3, p38, PI3K/Akt and MAPK/Erk. IFNτ reduced (p < 0.01) the mRNA expression (RT-PCR) and concentration (ELISA) of genes and proteins that mediate NETs formation in blood neutrophils including histones (H1, H2), neutrophil elastase (NE) and myeloperoxidase (MPO). However, the effects of IFNτ on these genes and proteins were eliminated in the presence of JAK3 or PI3K inhibitors. Immunocytochemistry study also showed strong MPO signal in the presence of JAK3 or PI3K inhibitors as compared to positive control (PC, IFNτ alone). The results indicate that IFNτ impairs NETs formation using JAK3 and PI3K and thus essential for successful implantation and establishment of pregnancy in cows.

Intrauterine administration of peripheral blood mononuclear cells activated by human chorionic gonadotropin in patients with repeated implantation failure: A meta-analysis.

The purpose of this study was to assess whether intrauterine administration of peripheral blood mononuclear cells (PBMCs) activated by human chorionic gonadotropin (hCG) could improve the pregnancy and live birth rates in women with repeated implantation failure (RIF), and whether the parameters of co-culture of hCG and PBMCs would affect the clinical outcomes. Six databases (PubMed, Ovid, Medline, NCBI, Cqvip and Wanfang) were searched up to October 2020 by two independent reviewers. Seven studies were included according to specific inclusion and exclusion criteria. A meta-analysis showed that the pregnancy and live birth rates were significantly increased in the case group compared with the control group (odds ratio [OR]: 3.43, 95 % confidence interval [CI]: 1.78-6.61; P = 0.0002 and OR: 2.79, 95 % CI: 1.09-7.15; P = 0.03), especially when hCG was cultured with PBMCs for 48 h or PBMCs administration was performed two or three days before embryo transfer (ET). Neither the dosage of the hCG co-cultured with PBMCs nor the mean concentration of the administered PBMCs appeared to influence the therapeutic efficiency. In conclusion, intrauterine administration of PBMCs co-cultured with hCG for 48 h, conducted two or three days before ET, could be an effective therapy for women experiencing RIF. Due to the limitations of sample size and quality of the included studies, further high-quality studies with large sample sizes are warranted to optimize the parameters of hCG and PBMC co-culture to help more RIF patients benefit from this therapy.

Unexplained recurrent implantation failures: Predictive factors of pregnancy and therapeutic management from a French multicentre study.

INTRODUCTION: Recurrent implantation failure is defined as the absence of pregnancy after at least three transfers of good-quality embryos after in vitro fecundation/intracytoplasic sperm injection. AIM: The aim of this study was to describe a multicentre cohort of women with unexplained RIF, to analyse the factors associated with clinical pregnancy and to evaluate the immunomodulatory therapies efficacy. METHODS: Women were consecutively recruited from university departments with unexplained RIF. RESULTS: Sixty-four women were enrolled with mean age 36 ± 3 years. The rates of clinical pregnancy in 64 women were compared in untreated and treated cycles and according to therapies used during the last prospectively followed embryo transfer. A clinical pregnancy after the transfer was noted in 56 % pregnancies on intralipids and in 50 % on prednisone, versus 5 % in untreated ones (p < 0.001). The 340 embryo transfers of these 64 women resulted in 68 clinical pregnancies and 18 live births. Clinical pregnancies were significantly more frequent in treated versus untreated embryo transfers (44 % vs 9 %; p < 0.001) with odds ratio at 8.13 (95 % CI 4.49-14.72, p < 0.0001). Cumulative pregnancy rates were higher for steroid-treated transfers than for untreated transfers when considering overall transfers before and after using steroids and also only those under steroids. Cumulative pregnancy rates were not different from steroid- and intralipid-treated embryo transfers CONCLUSIONS: In this multicentre study of women with unexplained RIF, use of immunomodulatory treatments before embryo transfer resulted in higher clinical pregnancy. Randomised, well-designed studies in well-defined population of RIF women are necessary to confirm our preliminary data.

Granzyme B-expressing γδ-T and NK cells as a predictor of clinical pregnancy failure in patients with unexplained repeated implantation failure.

The limited cytotoxicity of immune cells facilitates a successful establishment of pregnancy. However, the association between cytotoxic granules and unexplained repeated implantation failure (uRIF) remains unkown. Twenty-one fertile controls and 54 patients with uRIF were included in this study. The pregnancy outcomes were monitored at different gestational periods. The peripheral blood lymphocytes were detected using specific monoclonal antibodies by flow cytometry. The percentage of perforin+ (Pfr+), granzyme B+ (GrB+), or granulysin+ (Gnly+) lymphocytes was not significantly different among fertile controls, uRIF patients with successful pregnancy outcomes, and uRIF patients with pregnancy failure. The percentage of GrB+ γδ-T cells in lymphocytes was markedly higher in uRIF patients with implantation failure and clinical pregnancy failure than that in uRIF patients with a corresponding successful pregnancy outcome. A four-tier risk model showed that the risk of suffering clinical pregnancy failure in uRIF patients among high risk tier (83.3 %), normal risk tier (65.0 %) and low risk tier (39.1 %) was elevated by 2-4 fold compared with uRIF patients among lowest risk tier (20.0 %). In addition, the percentage of GrB+ NK cells in lymphocytes tended to decrease in uRIF patients with pregnancy failure. The AUC of the combined indicator with GrB+ γδ-T cells and GrB+ NK cells was increased than that of GrB+ γδ-T cells and GrB+ NK cells for predicting clinical pregnancy failure. In conclusion, the frequency of GrB-expressing γδ-T and NK cells in peripheral blood could serve as a predictor of clinical pregnancy failure in patients with uRIF.

Direct and Indirect endocrine-mediated suppression of human endometrial CD8+T cell cytotoxicity.

Regulation of endometrial (EM) CD8+T cells is essential for successful reproduction and protection against pathogens. Suppression of CD8+T cells is necessary for a tolerogenic environment that promotes implantation and pregnancy. However, the mechanisms regulating this process remain unclear. Sex hormones are known to control immune responses directly on immune cells and indirectly through the tissue environment. When the actions of estradiol (E2), progesterone (P) and TGFß on EM CD8+T cells were evaluated, cytotoxic activity, perforin and granzymes were directly suppressed by E2 and TGFß but not P. Moreover, incubation of polarized EM epithelial cells with P, but not E2, increased TGFß secretion. These findings suggest that E2 acts directly on CD8+T cell to suppress cytotoxic activity while P acts indirectly through induction of TGFß production. Understanding the mechanisms involved in regulating endometrial CD8+T cells is essential for optimizing reproductive success and developing protective strategies against genital infections and gynecological cancers.

Kynurenine promotes the cytotoxicity of NK cells through aryl hydrocarbon receptor in early pregnancy.

During early pregnancy, decidual NK (dNK) cells play indispensable roles in many processes including the decidualization, the implantation, and the maintenance of immune tolerance. Abnormal cytotoxic activity of NK cells can cause recurrent spontaneous abortion (RSA), while the regulatory mechanism of NK cytotoxicity remains to be unclear. In this study, we found that kynurenine in decidua and villus was in a comparable level between patients with RSA and normal pregnancy women. However, the aryl hydrocarbon receptor (AhR) in decidual NK cells was significantly increased in RSA. Compared with AhR- NK cells, cytotoxic activity-related molecules (NKP30, NKP46, NKG2D, perforin, granzyme B and IFN-γ) was highly expressed in both AhR+ peripheral and decidual NK cells, and kynurenine stimulation promoted the expression of killer receptors and the cytoplasmic granules in an AhR-dependent manner. Stimulation with TNF-α, IL-ß and LPS upregulated the AhR expression in dNK cells in vitro. These results indicate that kyn/AhR signal enhances the cytotoxicity of NK cells, and increased expression of AhR may be an induction factor of RSA.