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Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform ...

Shenzhen Zhongshan Urology Hospital

Thyroid autoimmunity (TAI), which is defined as the presence of autoantibodies against thyroid peroxidase (TPO) and/or thyroglobulin (TG), is related to repeated implantation failure (RIF). It is reported that TAI was involved in reproductive failure not only through leading thyroid function abnormality, but it can also be accompanied with immune imbalance. Therefore, this study was designed to investigate the association of thyroid function, immune status and TAI in women with RIF. Blood samples were drawn from 72 women with RIF to evaluate the prevalence of TAI, the thyroid function, the absolute numbers and percentages of lymphocytes. The prevalence of thyroid function abnormality in RIF women with TAI was not significantly different from that in RIF women without TAI (c(2) = 0.484, p > 0.05). The absolute number and percentage of T cells, T helper (Th) cells, B cells and natural killer (NK) cells were not significantly different in RIF women with TAI compared to those without TAI (all p > 0.05). The percentage of T cytotoxicity (Tc) cells was significantly decreased in RIF women with TAI compared to those without TAI (p < 0.05). Meanwhile, Th/Tc ratio was significantly increased (p < 0.05). These results indicated that the decreased Tc percentage and increased Th/Tc ratio may be another influential factor of adverse pregnancy outcomes in RIF women with TAI.

Thyroid Autoimmunity is Associated with Decreased Cytotoxicity T Cells in Women with Repeated Implantation Failure.

Inhibitor of DNA-binding protein 3 (Id3) is required for tumor angiogenesis and regulatory T-cell generation. However, the involvement of Id3 in unexplained repeated implantation failure (RIF) and recurrent miscarriage (RM) remains poorly understood. Immunohistochemistry was used to identify Id3, CD34, CTLA-4, and FOXP3 in the endometrium taken from the women with RIF (n=16), RM (n=16) and matched controls (n=8). The images were acquired and analyzed by the Vectra automated quantitative pathology imaging system. Percentage of Id3 cells was significantly higher in the endometrium of women with RIF and RM compared with controls. The numbers of Id3 and CD34 vessels in the endometrium were positively correlated in control but not in RIF or RM. Percentages of CTLA-4 cells, but not FOXP3 cells, were significantly increased in the endometrium of RIF and RM women than those in controls. We found aberrant expressions of endometrial Id3 and CTLA-4 in peri-implantation endometrium of women with RIF and RM, suggesting the negative roles of these angiogenesis and immune tolerance markers involving in regulating endometrium receptivity.

Aberrant expressions of endometrial Id3 and CTLA-4 are associated with unexplained repeated implantation failure and recurrent miscarriage.

To investigate whether chronic endometritis (CE) affects the immune status of peripheral blood and endometrium in patients with recurrent reproductive failure (RRF).

Evaluation of peripheral and uterine immune status of chronic endometritis in patients with recurrent reproductive failure.

Polycystic ovary syndrome (PCOS) is described as a low-grade chronic inflammatory state. However, there are limited studies on the specific endometrial immune status of PCOS patients. Whether this endometrial immune cell pattern is intrinsic to PCOS or the consequence of PCOS-associated obesity is a subject of debate. This study retrospectively included one hundred women diagnosed with PCOS and ninety-five normal fertile controls, which further divided into four groups (normoweight PCOS; overweight PCOS; normoweight control; overweight control) based on body mass index. The percentages of endometrial CD68 macrophages (1.97 % vs. 1.17 %; P < 0.001), CD163 M2 macrophages (2.30 % vs. 1.83 %; P = 0.001), CD1a iDCs (0.044 % vs. 0.029 %; P = 0.002), CD83 mDCs (1.72 % vs. 1.07 %; P < 0.001) and CD8 T cells (2.82 % vs. 1.95 %; P < 0.001) were significantly higher in normoweight PCOS women than normoweight controls. The percentage of CD68 macrophages (2.09 % vs. 1.15 %; P < 0.001) was significantly higher in overweight PCOS women compared with overweight controls. In multivariant linear regression analysis, participants' PCOS status was the main predictors of endometrial CD68 macrophages, CD163 M2 macrophages, CD1a iDCs, CD83 mDCs and CD8 T cells in the whole study population. Additionally, in PCOS group, positive correlations were found between endometrial CD56 NK, CD163 M2 macrophages and QUICKI, indicating there was an association between endometrial immune cells and insulin resistance in PCOS women. Our study suggests that women with PCOS have altered endometrial immune cells, which may reflect a state of chronic low grade inflammation. The chronic inflammation, independent of obesity, may help understand the pathophysiologic mechanisms of intrinsic PCOS.

Evaluation of endometrial immune status of polycystic ovary syndrome.

To investigate whether endometrial T-bet (Th1 lineage-committed transcription factor)/GATA3 (Th2 lineage-committed transcription factor) ratio has predictive potential for embryo implantation in infertile women undergoing in vitro fertilization-embryo transfer (IVF-ET).

Predictive role of endometrial T-bet/GATA3 ratio during mid-luteal phase for live birth in patients undergoing in vitro fertilization: A retrospective observational study.

This study aimed to develop reference intervals (RIs) of endometrial immune cells in control infertile women during the mid-luteal phase, and compare with the proportion of endometrial immune cells in recurrent reproductive failure (RRF) patients. Endometrial tissue sections were obtained from 113 fertile women and 79 patients with RRF, including 40 patients who had suffered recurrent miscarriage (RM) and 39 patients with repeated implantation failure (RIF) during the mid-luteal phase of the menstrual cycle. Immunohistochemical staining and quantitative analysis of CD56, Foxp3, CD163, CD1a and CD8 cells were performed in endometriums. RIs of endometrial immune cells in control infertile women were as follows: CD56 uterine natural killer cells (uNK) cells, 1.785-8.712%, forkhead box P3 (Foxp3) Tregs, 0.041-0.154%, CD163 M2 macrophages, 0.298-1.492%, CD1a dendritic cells (DCs), 0.006-0.081% and CD8 T cells, 0.674-2.504%. Compared with control infertile women, the percentage of endometrial CD56 uNK cells, CD163 M2 macrophages, CD1a DCs and CD8 T cells were significantly increased in patients with RRF. Moreover, Foxp3 Tregs levels were decreased in patients with RRF, and were statistically significant only in patients with RM. In conclusion, the RIs of endometrial immune cells were established in control infertile women during the mid-luteal phase, and a disordered endometrial immune microenvironment was observed in patients with RRF. The RIs of endometrial immune cells may be of important clinical significance for the treatment of RRF.

Establishment of reference intervals of endometrial immune cells during the mid-luteal phase.

This study aimed to investigate whether maternal obesity affects the immune status of peripheral blood and endometrium in patients with recurrent reproductive failure classified according to their body mass index (BMI). A total of 228 repeated implantation failure (RIF) and 266 recurrent miscarriage (RM) patients were enrolled in the study and further subdivided into three groups according to their BMI: (i) normal weight (18.5≤ BMI <23); (ii) overweight (23≤ BMI <25); and (iii) obese (BMI ≥25). Peripheral blood and endometrium samples were collected in the mid-luteal phase before IVF treatment or natural pregnancy. Peripheral immunocytes were analyzed by flow cytometry, while uterine immune cells were subjected to immunohistochemistry. In RM patients, significantly increased peripheral helper T cells and decreased cytotoxic T cells, NK cells were observed in the obese group compared with the normal-weight group. Meanwhile, in the endometrium, the percentage of NK cell, macrophage cell, M2 macrophage cell, and Treg cell significantly reduced with increased BMI in RIF patients, and the percentage of NK cell and M2 macrophage cell significantly decreased with increased BMI in RM patients. In conclusion, obesity may cause endometrial immune disorder in recurrent reproductive failure women, but was only associated with the peripheral immune change in RM patients.

Impact of body mass index on peripheral and uterine immune status in the window of implantation in patients with recurrent reproductive failure.

Is the ratio of endometrial T-box expressed in T cell (T-bet) and GATA-binding protein 3 (GATA3) changed in patients with recurrent miscarriage (RM) compared to fertile controls?

Comparing the peri-implantation endometrial T-bet/GATA3 ratio between control fertile women and patients with recurrent miscarriage: establishment and application of a reference range.

RM is a common clinical disease in reproduction, affecting approximately 1%-3% of women worldwide. Previous studies have shown the role of peripheral blood γδ-T cells during physiological pregnancy. However, the relationship between the immune status of peripheral blood γδ-T cells and RM is still not well defined.

Cong Chen

Shenzhen Key Laboratory of Reproductive Immunology for Peri-implantation,Shenzhen Zhongshan Institute for Reproduction and Genetics,Fertility Center,Shenzhen Key Laboratory of Reproductive Immunology for Peri-implantation, Shenzhen Zhongshan Institute for Reproduction and Genetics, Fertility Center

Shenzhen Zhongshan Urology Hospital

Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis