Endometrial NK Cells Research Articles

Endometrial NK cells in repeated implantation failure – quantity and functional markers

BACKGROUND: Alteration in the composition and function of endometrial immune cells, in particular NK cells, are associated with implantation and placentation pathology, which is considered as one of the causes of reproductive losses. However, data regarding abnormalities in the number and activity of NK cells in repeated implantation failures, including depending on the type of infertility, remain ambiguous.
 AIM: The aim of this study was to evaluate the number of CD56+ and CD16+ cells and the area of expression of CD107a and NKG2D markers in the endometrium of patients with repeated implantation failure (RIF).
 MATERIALS AND METHODS: This prospective comparative study included patients with RIF (main group I, n = 47), who were divided into two subgroups: Ia, patients with primary infertility (n = 29); Ib, patients with secondary infertility (n = 18). Comparison group II included patients with a history of effective ART programs (n = 17). Control group III included healthy fertile women without a history of reproductive loss (n = 12). Endometrial biopsies were obtained on days 19–23 of the menstrual cycle. The expression of CD56+, CD16+, CD107a and NKG2D was assessed by immunohistochemistry.
 RESULTS: In the endometrium of patients in groups I and II, as compared to the control group, the number of CD56+ cells was significantly increased (p 0.001). In patients of subgroup Ia, when compared to the control group, we verified an increase in the number of CD16+ cells (p 0.05) and a decrease in the expression of CD107a (p 0.05). In patients in groups I and II, a negative correlation was revealed between the number of CD56+ and CD16+ cells and the number of pregnancies in history (rs = –0.30 and rs = –0.34, p 0.05), while a positive correlation was found between the expressions of CD56+ and CD107a (rs = 0.66 and rs = 0.75, p 0.05). In patients in group II, a positive correlation was revealed between the expressions of CD16+ and CD107a (rs = 0.75, p 0.05). In the endometrial stroma, CD107a expression increased significantly in patients in group I (p 0.05), while NKG2D expression increased in groups II and III (p 0.01, p 0.05) from the early to the middle stage of the secretion phase. In patients of group II, a positive correlation was established between the expressions of CD56+ and NKG2D (rs = 0.68, p 0.05).
 CONCLUSIONS: In the endometrium of patients with primary infertility and RIF, the number of CD56+ and CD16+ cells is increased with a decrease in the expression of their activation marker CD107a, which may be a potential mechanism for impaired implantation. Further studies of the immune profile of the endometrium may help to personalize diagnostic and therapeutic approaches to management of patients with RIF and to increase the chances of pregnancy in ART programs.

Changes in the immunophenotype of endometrium during implantation window receptivity formation in healthy fertile women

IntroductionEndometrial receptivity is largely determined by the immunophenotype of endometrium, especially uterine NK-cells (uNK). Immune component is directly involved in the formation of favourable microenvironment for the blastocyst implantation and placenta formation, but the way it changes during the maturation of endometrial tissue in healthy fertile women is still underexplored. MethodsThe endometrium was collected from 47 healthy oocyte donors after controlled ovarian stimulation: 23 women on the day of oocytes retrieval (OR) and 24 women on the term of implantation window (IW). The OR group was analysed, published previously and used as a comparison group to show the dynamic of changes.Isolated endometrial lymphocytes and peripheral blood samples were stained with monoclonal antibodies and analysed according to the three-color flow cytometry protocol. ResultsThe proportion of NK-cells (CD3−CD56+) in endometrium grew significantly in the implantation window compared to the oocytes retrieval day. NK-cells acquired a more differentiated phenotype from the day of OR until IW: the expression of CD8 and CD158a significantly increased, while the expression of HLA-DR significantly decreased. Significant correlations between peripheral blood and endometrial NK-cells were found in CD8 expression during OR and IW, CD335(p46)neg and CD335(p46)++ subsets during IW term. DiscussionImmunophenotype of receptive endometrium forms due to the accumulation of uNK-cells, which actively proliferate, become mature, differentiative, and ready to meet the embryo.Endometrial immunophenotype is peculiar and specific but not autonomic and isolated.Differentiation (CD8 on NK-cells), and activity (p46 on NK-cells) of peripheral blood lymphocytes is reflected in endometrial lymphocytes profile, and therefore the research of peripheral blood immunophenotype is relevant.

The effect of moderate aerobic exercise on the composition of endometrial NK cells in women with idiopathic Recurrent Pregnancy Loss

The effect of moderate aerobic exercise on the composition of endometrial NK cells in women with idiopathic Recurrent Pregnancy Loss

How platelet-rich plasma (PRP) intra-uterine injection improve endometrial receptivity of intrauterine adhesions in women: A time-series-based self-controlled study

Platelet-rich plasma (PRP) treatment proven to improve fertility outcomes in patients with a poor endometrial environment. However, the mechanism is not yet clear. In this study, we recruited 6 patients with infertility due to IUA and 6 normal control women. The subjects in the IUA group collected samples before and after PRP treatment. Endometrial receptivity was improved after PRP treatment. After PRP treatment, the endometrial NK cells, CD8 T cells and Th1 cells were significantly lower than those before treatment. Functional enrichment analysis suggested that the effects of changes in microbial composition played an important role in changes in the endometrial immune environment. Among them, the most significant difference was Bacillus. Our self-controlled cohort in this study can fully describe the detailed mechanism by which PRP treatment improves the endometrial environment.

Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares—A Preliminary Study

Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cytometry with anti-CD4, -CD25 and -FOXP3 and anti-NKp46 and -CD3 antibodies was used to determine the populations of Tregs and NK cells, respectively. The concentration of progesterone was measured with chemiluminescence immunoassay. The results were analyzed with paired Student t tests. The mean percentage of endometrial CD4+FOXP3+ Tregs was 13.7 ± 6.2% in diestrus and 14.5 ± 5.9% in estrus, while the mean percentage of endometrial CD4+FOXP3+CD25+ Tregs changed from 3.6 ± 2.1% in diestrus to 2 ± 2% in estrus (p = 0.0947). The mean proportion of CD3-NKp46+ lymphocytes in the endometrium was not significantly different, with 6 ± 1% in estrus and 6.5 ± 1.4% in diestrus. There was a large variation in the percentage of NK cells between mares of 2.1-12.7%. This study showed, for the first time, the presence of CD4+FOXP3+CD25+ Tregs and CD3-NKp46+ NK cells in the endometrium of non-pregnant cycling mares. The percentage of Tregs, and to a greater extent NK cells, showed large fluctuations between mares. Both Tregs and NK cells might be important for the preparation of the endometrium for semen deposition and pregnancy; however, further research is required.

Uterine Natural Killer Cells: A Rising Star in Human Pregnancy Regulation.

Uterine natural killer (uNK) cells are an immune subset located in the uterus. uNK cells have distinct tissue-specific characteristics compared to their counterparts in peripheral blood and lymphoid organs. Based on their location and the pregnancy status of the host, uNK cells are classified as endometrial NK (eNK) cells or decidua NK (dNK) cells. uNK cells are important in protecting the host from pathogen invasion and contribute to a series of physiological processes that affect successful pregnancy, including uterine spiral artery remodeling, fetal development, and immunity tolerance. Abnormal alterations in uNK cell numbers and/or impaired function may cause pregnancy complications, such as recurrent miscarriage, preeclampsia, or even infertility. In this review, we introduce recent advances in human uNK cell research under normal physiological or pathological conditions, and summarize their unique influences on the process of pregnancy complications or uterine diseases. Finally, we propose the potential clinical use of uNK cells as a novel cellular immunotherapeutic approach for reproductive disorders.

S11.1: Endometrial NK and plasma cells in infertile women with RPL, RIF and chronic endometritis

S11.1: Endometrial NK and plasma cells in infertile women with RPL, RIF and chronic endometritis

CD200S-positive granulated lymphoid cells in endometrium appear to be CD56-positive uterine NK cells

The eutopic secretory phase endometrium in endometriosis overproduces and releases a soluble immunosuppressive CD200 molecule (CD200L) and is populated by stromal cells that contain a truncated CD200 (CD200S) that promotes a proinflammatory environment. The CD200S+ cell population persists when pregnancy occurs and are abundant in the early pregnancy decidua of women with missed abortion. In the present study, CD200S+, CD56+, and CD68+ cells were enumerated in formalin-fixed paraffin-embedded tissue sections from women with endometriosis and non-endometriosis controls. CD200S+ cells were more numerous than CD68+ macrophages and were similar in number and location to CD56bright endometrial NK cells. In some endometria, there was an additional population of CD200S- CD56+ NK cells. In ectopic endometrial peritoneal deposits and in ectopic myometrial deposits (adenomyosis), CD200S+ cells were less frequent, consistent with the known paucity of CD56+ NK cells in sites of ectopic deposits. CD200S+ cell frequency was greater in stroma surrounding the smaller ectopic cystic deposits. Dual immunofluorescent antibody staining confirmed CD200S+ cells were CD56+ NK cells. CD200S+ NK cell frequency may be greater in endometriosis patients’ endometrium and may affect embryo survival in early pregnancy. In our opinion, regulation of alternative splicing that results in CD200S rather than CD200L may provide new diagnostic and therapeutic options for women with endometriosis.

Origin of human uterine natural killer cells

Abstract Uterine NK cells (uNK) are a distinct immune population which influence pregnancy outcome. While murine studies have suggested that tissue-resident uNKs predominate in virgin endometrium, the origin of uNKs in humans is unknown. To define subsets of uterine NKs in humans and determine their developmental origin, we performed single-cell RNA-sequencing on CD56+ uNKs sorted from endometrial biopsies of 4 healthy controls (HCs) (17,146 cells) and 5 uterus transplant (UTx) recipients (30,351 cells). SNP polymorphisms and HLA alleles were used to identify cells of donor (tissue-resident) or recipient (peripheral) origin in UTx recipients. Developmental trajectories were built with Monocle v.3.0. Ten major clusters were identified in HCs, with 48% of endometrial NK cells (eNKs) occupying 3 clusters (eNK1–3) that shared gene signatures with known human decidual NK cell subsets (dNK1–3). Pseudotime analysis revealed that the eNK1–3 clusters arose from proliferating subsets and were less mature than cytotoxic uNKs expressing ZEB2, TBX21, and FCGR3A (CD16). uNKs from UTx recipients were similarly distributed across the clusters, with a trend towards fewer cells in the FCGR3A-expressing mature subset. Notably, recipient-derived uNKs dominated all uNK subsets within four months post-transplant but became less frequent in UTx recipients >1 year after transplant (95% vs. 72%; early vs. late). The proportion of donor-derived to recipient-derived cells remained constant across subsets in all UTx recipients. In contrast to mouse models, our study suggests that human uNKs derive primarily from blood-borne peripheral immigrants and that environmental factors influence uNK differentiation more than genotype or developmental origin.

Co-expression of NKp46 with activating or inhibitory receptors on, and cytokine production by, uterine endometrial NK cells in recurrent pregnancy loss

NKp46 (CD335) is one of the activating receptors expressed on NK cells and its expression is decreased in patients with reproductive failure. However, the reasons remain unknown. In this study, we aimed to clarify the significance of decreased NKp46 expression in reproductive failure. Uterine endometrial samples collected from 39 patients with recurrent pregnancy loss (RPL) were assigned to high- or low-risk groups based on an 18 % ratio of CD16+/CD56dim NK cells in uterine endometrial NK (uNK) cells. We analyzed the expression of NKp46 and other activating or inhibitory receptors on, and intracellular cytokine production by NK cells using multicolor flow cytometry. The numbers of NKp46+/CD16− NK, NKp46+/NKG2C− NK, IL-4+/CD56+ NK, and IL-10+/CD56+ NK cells were significantly decreased, whereas that TNF-α+/CD56+ NK cells was significantly increased in the high-risk group, when compared with the low-risk group (P < 0.05 for all). The ratios of TNF-α/IL-4, IFN-γ/IL4, TNF-α/IL-10, and IFN-γ/IL10 cytokine production in uNK cells were significantly increased in the high-risk when compared with the low-risk group (P < 0.05, for all). It is suggested that low expression of activating receptors on NKp46 uNK cells is more prevalent in high-risk women.

A semen-based stimulation method to analyze cytokine production by uterine CD56bright natural killer cells in women with recurrent pregnancy loss

Cytokine secretion by NK cells is abnormal in some women with recurrent pregnancy loss (RPL). Cytokine production is usually evaluated after stimulation with PMA and ionomycin. However, stimulation of uterine NK cells with semen corresponds more closely to physiological conditions at the time of conception. As seminal plasma has immunomodulatory properties, we aimed to elucidate compatibility between uterine NK cells and semen. Endometrial samples were stimulated with PMA/ionomycin, semen, seminal plasma, or spermatozoa. Thereafter, cytokine production by NK (CD56bright) cells was evaluated using flow cytometry and compared between women with and without a history of RPL associated with abnormal NK cell distribution in the endometrium or unexplained RPL. The ratios (%) of NK cells producing IFN-γ and TNF-α (NK1 phenotype), IL-4 (NK1/NK2 phenotype), and IL-10 (NK1/NKr1 phenotype) were significantly lower after stimulation with semen than with PMA/ionomycin (P < 0.01). After exposure to semen, ratios (%) of NK cells producing IL-4 and IL-10 in patients with unexplained RPL were significantly lower (P < 0.05), whereas those of NK1/NK2 and NK1/NKr1 were significantly higher (P < 0.01) than those in controls. The shift of endometrial NK cells to the NK2 phenotype was more pronounced when stimulated by semen than by PMA/ionomycin. However, a semen-induced shift to NK1 in women with unexplained RPL could induce miscarriage. Couple-specific immunological compatibility tests through semen stimulation in vitro might provide important information to avoid RPL.

Normalization of endometrial histopathology and endometrial NK cells concentration predict successful pregnancy in repeated implantation failure.

Objective:The primary objective was to establish the endometrial predictors of clinical pregnancy in a population of repeated implantation failure with oocyte donation after specific endometrial treatment. The secondary one was to evaluate reproduction outcomes in terms of Implantation rate (IR), Clinical pregnancy (CP), Live birth delivery rate (LBDR) and Prematurity, in relation to normalization or no-normalization of the predictors.Methods:66 patients were assigned to the study. We ran a Pipelle endometrial biopsy to investigate the endometrium lymphocyte population by Flow Cytometry and abnormal/normal patterns by histopathology in pre/post-treatment. We employed the binary logistic regression model to identify the predictors for CP. For the secondary objective, we assessed the clinical outcomes in function to the normalization or no normalization in post-treatment. Results:Endometrial histopathology and endometrial NK cell counts resulted in CP predictors (Wald chi2 test (p=0.044 and 0.001)), respectively. We had a higher IR, CP and LBDR when both predictors were normalized in comparison with no normalization (p<0.001). There was a high percentage of prematurity in both normalized vs. non-normalized groups (34.4% (11/32) and 71.43% (5/7), respectively) without significant differences.Conclusion:Endometrial histopathology and endometrial NK cell counts showed that they are valid predictors of pregnancy outcome in repeated implantation failure after treatment. In post-treatment, the pregnancy outcomes were significantly higher in the presence of both normalized predictors. Pregnancy rates were zero in the no-normalization of both predictors. There was a high percentage of prematurity in both groups.

Endometrial NK and plasma cells in infertile women

Endometrial NK and plasma cells in infertile women

P93: Endometrial NK and plasma cells in infertile women

American Journal of Reproductive ImmunologyVolume 81, Issue S1 p. 80-80 ABSTRACTS P93: Endometrial NK and plasma cells in infertile women First published: 31 May 2019 https://doi.org/10.1111/aji.91_13120Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume81, IssueS1Special Issue: The 39th Annual Meeting of the American Society for Reproductive Immunology, 12-15 June 2019, Grand Rapids, Michigan, USAMay 2019Pages 80-80 RelatedInformation

Decidual stromal cells promote the differentiation of CD56bright CD16- NK cells by secreting IL-24 in early pregnancy.

Decidual stromal cells (DSCs) are important origins of cytokines to modulate maternal-fetal immunotolerance and provide a feasible environment for embryo implantation and development. Interleukin (IL)-24 is a multifunctional cancer killing cytokine and a pleiotropic immunoregulator with complex potency according to tissue or cell types. Its role in establishment and maintenance of normal pregnancy is largely unknown. The aim of our study was to investigate the function and significance of IL-24 and its receptor in the coordination between DSCs and natural killer cells (NK) in early pregnancy. The levels of IL-24 in DSC, endometrial stromal cell (ESC), peripheral blood NK cells(pNK), or decidual NK cells(dNK) culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA), and the levels of IL-24 receptors were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry assays. The effect of IL-24 on the functions of decidual NK cells was analyzed by flow cytometry assays in vitro. The concentration of IL-24 in culture supernatant of DSCs was significantly higher than that of ESCs. Both eNK (endometrial NK cells) and dNK highly expressed IL-24 receptors (IL-20R1 and IL-22R1), especially on CD56dim eNK. However, there were extremely low levels of IL-20R1 and IL-22R1 on pNK. Recombinant human IL-24 or DSCs-secreted IL-24 downregulated the levels of CD16, Granzyme B, perforin, and interferon (IFN)-γ and upregulated the levels of inhibitory receptors killer-cell immunoglobulin-like receptor (KIR)2DL1 and KIR3DL1, or immunotolerant or angiogenic cytokines (eg, transforming growth factor (TGF)-β, IL-10, and IL-8), and elevated the percentage of CD56bright CD16- dNK in vitro. These data suggest that DSCs promote the differentiation of CD56bright CD16- NK with high levels of inhibitory receptors, immunotolerant, and angiogenic cytokines by secreting IL-24 during decidualization in early pregnancy.

Selective expansion and CMV-dependency in pregnancy trained human endometrial NK cells

Selective expansion and CMV-dependency in pregnancy trained human endometrial NK cells

The effect of prednisolone on endometrial uterine NK cell concentrations and pregnancy outcome in women with reproductive failure. A retrospective cohort study

This retrospective study of prospectively collected data examines the effect of prednisolone therapy on raised uterine Natural Killer cell (uNK) concentrations and pregnancy outcomes in women with recurrent miscarriage (RM) and recurrent implantation failure (RIF) after IVF/ICSI treatment. 136 women diagnosed with RRF who had a timed midluteal endometrial biopsy taken for uNK cell analysis were included. Women with high uNK cell concentrations (n = 45) were treated with prednisolone (10 mg/day) for one month, after which a second biopsy was taken for repeat uNK cell analysis. Women for whom prednisolone caused a decrease in uNK cell concentrations continued on prednisolone until 12 weeks of pregnancy. Pregnancy outcomes (live birth, miscarriage and implantation rates) and pregnancy complications were compared for women who received prednisolone and those who did not. Results showed that the prevalence of high uNK cells was 33.1%. Prednisolone significantly decreased the uNK cell concentration (P < 0.001), however reduction to normal limits was achieved in only 48.3% of patients. There was no difference in any of the pregnancy outcomes or complications between women who had received prednisolone and those who had not. In conclusion, this study showed a relatively high prevalence of raised uNK cells in women with recurrent reproductive failure and confirmed the effect of prednisolone on reducing uNK cell concentrations. We found however no evidence for a significant beneficial effect for prednisolone therapy on pregnancy outcomes. Until the results of an adequately powered RCT become available however, these findings should be considered preliminary.

Various expression of CD16 on uterine endometrial NK cells

Various expression of CD16 on uterine endometrial NK cells

The co-expression of activating and inhibitory receptor and cytokines production for uterine endometrial NK cells

The co-expression of activating and inhibitory receptor and cytokines production for uterine endometrial NK cells

THE STUDY OF IMMUNOHISTOCHEMICAL EXPRESSION OF CERTAIN PRO- AND ANTIAPOPTOTHIC FACTORS IN ENDOMETRY OF PATIENTS WITH ADENOMIOSIS

Objective: to study the immunohistochemical expression of proand anti-apoptotic factors — FasL(CD95), TNF-a, p65 NFkB, and granzyme B in the endometrium of patients with adenomyosis. Material and Methods. The endometrial samples, taken during the proliferative phase of the cycle, while carrying out paypel biopsy and hysteroscopy in 41 women aged 37 to 48 years with adenomyosis. The comparison group consisted of 25 women with infertility. The study of TNF-a, Granzym B, NFkB immunohistochemical expression in the development and progression of adenomyosis was performed using monoclonal antibodies. The results of reaction were evaluated by semiquantitative method. Statistical analysis was performed using the software package Biostat Apps MicrosoftR Excel full-featured office Microsoft Office 2010 package. Results. In the comparison group, the expression of TNF-a and NFkB in endometrium was not evident. In the group of patients with adenomyosis, a marked expression of these markers in epithelial and stromal cells was observed. Granzym B expression was detected in the cytoplasm of leucocytes of the stroma of the endometrial functional layer with no statistically significant differences between the groups. Conclusion. Presumably, given the expression peaks of both TNF-a, p65 NFkB, the observed in adenomyosis increase (compared to normal) in expression of these molecules does not mean readiness of epithelial cells of the endometrial functional layer to apoptosis, as is the norm, upon the occurrence of menstruation, but it means readiness of these cells to proliferate. The absence of statistically significant differences in the number of CD56-positive endometrial stromal granulocytes and endometrial NK-cells, determined by Granzym B expression in these cells, is also an indirect evidence of a low level of readiness to apoptosis. And statistically significantly less expression of FasL(CD95) in women with adenomyosis. That is, TNF-a and NFkB at adenomyosis, probably, play the role of cell survival factors.