Impairment of Decidualization of Endometrial Stromal Cells by hsa-miR-375 Through NOX4 Targeting.

Decidualization of the endometrial stromal cells (ESCs) is essential for successful embryo implantation. It involves the transformation of fibroblastic cells into epithelial-like cells that secrete cytokines, growth factors, and proteins necessary for implantation. Previous studies have revealed altered expression of miR-375 in the endometrium of patients with recurrent implantation failure and the ectopic stromal cells of patients with endometriosis. However, the exact molecular mechanisms, particularly the role of microRNAs (miRNAs) in the regulation of decidualization, remain elusive. In this study, we investigated whether decidualization is affected by miR-375 and its potential target(s). The findings demonstrated the downregulation of the expression of miR-375 in the secretory phase compared to its expression in the proliferative phase of the endometrium in normal donors. In contrast, it was upregulated in the secretory phase of the endometrium in infertility patients. Furthermore, during decidualization of ESCs in vitro, overexpression of miR-375 significantly reduced the transcript-level expression of forkhead box protein O1 (FOXO1), prolactin (PRL), and insulin-like growth factor binding protein-1 (IGFBP1), the well-known decidual cell markers. Overexpression of miR-375 also resulted in reduced decidualization-derived intracellular and mitochondrial reactive oxygen species (ROS) levels. Using the luciferase assay, we confirmed that NADPH oxidase 4 (NOX4) is a direct target of miR-375. Collectively, the study showed that the miR-375-mediated NOX4 downregulation reduced ROS production and attenuated the decidualization of ESCs. It provides evidence that miR-375 is a negative regulator of decidualization and could serve as a potential target for combating infertility.

Comparison of the Automated Fluorescent Immunoassay System With Roche Elecsys and Beckman Coulter Access 2 Assays for Anti-Müllerian Hormone Measurement.

BACKGROUND: Since 2017, automated assays have been used in most clinical laboratories for anti-Müllerian hormone (AMH) level measurement. We evaluated the analytical performance of the newly developed automated fluorescent immunoassay system (AFIAS) AMH assay (Boditech Med, Gangwon-do, Korea) in comparison with the Roche Elecsys and Beckman Coulter Access 2 AMH assays. METHODS: Analytical performance of the AFIAS AMH assay was assessed in terms of linearity, repeatability, and within-laboratory precision (CV%) using human recombinant AMH samples according to the Clinical and Laboratory Standards Institute (CLSI) guidelines EP05 and EP06. Using 293 serum samples collected from an infertility clinic, the AMH levels were compared across AFIAS, Elecsys, and Access 2 AMH assays according to the CLSI EP09 guidelines. RESULTS: The AFIAS AMH assay results were linear across the measurement range of 0.420-72.386 pmol/L AMH, with repeatability of 6.341%. CV% of the AFIAS AMH assay for three levels of control, 1.786, 7.143, and 56.857 pmol/L, were 5.801%, 5.714%, and 6.228%, respectively. The results of the three AMH assays showed strong correlation: AFIAS and Elecsys [slope, 1.055 (95% confidence interval (CI), 1.022-1.088) and Spearman's rho, 0.978 (95% CI, 0.973-0.983)], Elecsys and Access 2 [slope, 0.813 (95% CI, 0.791-0.834) and Spearman's rho, 0.986 (95% CI, 0.983-0.989)], and AFIAS and Access 2 [slope, 0.836 (95% CI, 0.821-0.853) and Spearman's rho, 0.984 (95% CI, 0.980-0.988)]. CONCLUSIONS: The AFIAS AMH assay may be an alternative to the Roche Elecsys and Beckman Coulter Access 2 AMH assays.

Maternal and fetal safety of intravenous immunoglobulin in women with reproductive failure.

PROBLEM: Intravenous immunoglobulin G (IVIG) is an emerging regimen for women with reproductive failures (RF) during- or pre-pregnancy who have aberrant cellular immune reactions. Studies investigating teratogenicity of IVIG have been limited. Herein, we evaluated the fetal teratogenicity of IVIG and IVIG-related obstetric complications. METHOD OF STUDY: Women who used IVIG during pregnancy due to RF with cellular immune aberrances were enrolled from four medical centers in Korea. The pregnancy outcomes were collected. RESULTS: A total of 370 RF women who used IVIG during their pregnancy were enrolled. Most of the patients started the IVIG therapy before 12 weeks of gestation and 229 women continued IVIG treatment beyond 12 weeks of gestation. The mean age of the subjects was 34.8 years and the mean total dosage of IVIG was 125.3 g. A total of 307 women had livebirths and six of them were twins. Of 301 singleton livebirths, obstetric complications were developed as follows: preterm births (12.0%), gestational diabetes (7.0%), preeclampsia (4.0%), placental abruption (1.3%), placenta previa (4.3%), and placenta accrete (1.7%). Total six cases (1.99%) had major fetal anomalies in livebirths. The incidence of birth defects is similar to those of the general population in Korea and the previous report in infertile women. No IVIG -related viral contamination was noted. CONCLUSION: IVIG use during pregnancy did not increase obstetric complications and fetal teratogenicity. This study can be an evidence of maternal and fetal safety of IVIG administration during pregnancy.

Menopause is an inflection point of age-related immune changes in women.

Elevated proinflammatory cytokines in postmenopausal women is considered as one of the causes increasing the incidence of chronic inflammatory diseases. However, the details of postmenopausal immune changes have not yet been fully revealed. Thus, we investigated age-related immune changes in women and compared immune responses in postmenopausal and reproductive-age women. A total of 34 postmenopausal women and 91 reproductive-age women were included in the study. After isolating peripheral blood mononuclear cells, analysis of immunophenotypes and intracellular cytokine profiles were done. The proportion of natural killer (NK) cells was significantly higher, and the ratio of TNF-α- to IL-10-producing CD3+CD4 + T cells (Th1 to Th2) and the ratio of Th17 cells to CD4+CD25+Foxp3+ regulatory T (Treg) cells (Th17 to Treg) were higher, in postmenopausal women than in reproductive-age women. The Treg cell proportion was negatively correlated with the Th1 and Th2 cell proportions in reproductive-age women but not in postmenopausal women. As age increased, the proportion of Tregs was increased in reproductive-age women (r = 0.302, p = 0.004), whereas the proportion of Th1 cells was increased in postmenopausal women (r = 0.466, p = 0.005). FSH levels showed a positive correlation with Fopx3+ T cell and Treg cell (p = 0.04, 0.053, respectively), whereas Th17/Treg ratio and Th1 cell showed negative correlation with FSH.(p = 0.045, 0.024, respectively). In conclusion, postmenopausal women have higher proinflammatory immune statuses, as demonstrated by increased proportions of NK, Th1, and Th17 cells, altered correlations among NK and T cell subsets, and compromised balances between effector T cell subsets.

COVID-19 and immunomodulation treatment for women with reproductive failures.

COVID-19 pandemic is affecting various areas of health care, including human reproduction. Many women with reproductive failures, during the peri-implantation period and pregnancy, are on the immunotherapy using immune modulators and immunosuppressant due to underlying autoimmune diseases, cellular immune dysfunction, and rheumatic conditions. Many questions have been raised for women with immunotherapy during the COVID-19 pandemic, including infection susceptibility, how to manage women with an increased risk of and active COVID-19 infection. SARS-CoV-2 is a novel virus, and not enough information exists. Yet, we aim to review the data from previous coronavirus outbreaks and current COVID-19 and provide interim guidelines for immunotherapy in women with reproductive failures.

Effects of MTHFR C677T polymorphism on vitamin D, homocysteine and natural killer cell cytotoxicity in women with recurrent pregnancy losses.

STUDY QUESTION: Is there any relationship between vitamin D [25 (OH) vitamin D], total plasma homocysteine and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in women with recurrent pregnancy losses (RPL)? SUMMARY ANSWER: Women with MTHFR 677TT (homozygous mutation, TT) genotype have significantly lower vitamin D levels, higher homocysteine and natural killer (NK) cell cytotoxicities than those of women with MTHFR 677CC (wild type, CC) and 677CT (heterozygous mutation, CT) genotypes. WHAT IS KNOWN ALREADY: Vitamin D insufficiency, MTHFR C677T polymorphism and hyperhomocysteinemia have been reported as risk factors for RPL. However, the relationship between these risk factors is not known in this population. STUDY DESIGN, SIZE, DURATION: This is a retrospective cross-sectional study, including 837 women with RPL, who were enrolled in Reproductive Medicine and Immunology, Chicago Medical School, between 2012 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with two or more RPL prior to 20 weeks of gestation were included. To investigate whether the MTHFR C677T polymorphism affects the levels of homocysteine and vitamin D as well as immune parameters in women with RPL, biochemical data, such as plasma total homocysteine and serum vitamin D levels, and immune parameters, including NK cell cytotoxicity, were analyzed by MTHFR C677T genotype (CC, CT and TT). MAIN RESULTS AND THE ROLE OF CHANCE: The serum level of vitamin D in TT was significantly lower when compared with those of CT (P = 0.001) and CC (P = 0.003), while the level of homocysteine in TT was significantly higher than those in CT (P = 0.01) and CC (P = 0.01). NK cytotoxicity in TT was significantly higher than that of CC (P = 0.04) but not CT (P = 0.09). There was a significant negative correlation between the levels of vitamin D and homocysteine in TT (r = -0.357, P < 0.01). In multivariate analysis, vitamin D insufficiency (<30 ng/ml) was an independent risk factor for hyperhomocysteinemia (adjusted odds ratio 1.89, 95% CI 1.41-2.52). LIMITATIONS, REASONS FOR CAUTION: The study was retrospective and included only women with RPL but not healthy fertile controls. In addition, folic acid, vitamin B6 and B12 intake, which could modify the level of homocysteine and vitamin D, were not investigated. Thus, a considerable part of women might have folic acid and vitamin D supplementation and prenatal vitamin pills, and there are probable confounders in this study associated with unrestricted vitamin supplementation. Therefore, the findings should be carefully interpreted and applied to RPL women with MTHFR gene polymorphism. WIDER IMPLICATIONS OF THE FINDINGS: The findings attained in this analysis regarding the MTHFR polymorphism and its relationship with vitamin D, homocysteine and NK cytotoxicity may aid in uncovering the underlying etiology and mechanism for RPL. The study highlights an interplay between nutrition and immune responses in RPL. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this study. None of the authors have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

SIRT1 negatively regulates invasive and angiogenic activities of the extravillous trophoblast.

PROBLEM: Dysregulation of extravillous trophoblast (EVT) invasion leads to pregnancy complications, such as pre-eclampsia, fetal growth restriction, and placenta accreta. The aim of this study was to explore the role of SIRT1 in EVT invasion and its underlying mechanism. METHOD OF STUDY: SIRT1-specific siRNA was transfected into Swan 71 cells, an immortalized first trimester trophoblast cell line. The Boyden chamber invasion assay, the scratch wound healing assay, and cell proliferation assay were performed. The expression levels of epithelial-to-mesenchymal transition (EMT) markers, matrix metalloproteinase-2 (MMP-2), MMP-9, p-Akt, Akt, p-p38MAPK, p38MAPK, p-ERK, ERK, p-JNK, JNK, Fas, and Fas ligand (FasL) were examined by western blot. Tube formation assay was conducted by using Matrigel. RESULTS: SIRT1 knockdown by siRNA significantly enhanced invasion and migration as well as the expression of MMP-2, MMP-9, and EMT markers in Swan 71 cells, but reduced proliferation. The effects of SIRT1 knockdown on invasion, migration, proliferation, and endothelial-like tube formation in Swan 71 cells were reversely regulated by blockade of Akt and p38MAPK signaling. In addition, SIRT1 knockdown markedly promoted colocalization of Swan 71 cells to human umbilical vein endothelial cell (HUVEC) networks and induced reduction in Fas and enhancement of FasL. Conditioned media of SIRT1 knockdown-Swan 71 cells caused reduction in cell proliferation and augmentation of cytotoxicity along with increased Fas expression in HUVECs. CONCLUSION: Our results suggest that SIRT1 may be associated with placental development by controlling EVT invasion and spiral artery remodeling via modulation of EMT, MMP-2, MMP-9, Akt/p38MAPK signaling, and Fas/FasL.

1,25-Dihydroxy-vitamin D3 regulates NK-cell cytotoxicity, cytokine secretion, and degranulation in women with recurrent pregnancy losses.

Vitamin D has a pivotal role in regulating immune responses by promoting Th2 immune responses and suppressing Th1 responses. Propensities to a Th1 immune response and increased NK-cell levels and cytotoxicity have been reported in women with recurrent pregnancy losses (RPL). In women with RPL, vitamin D deficiency is prevalent; however, the effect of vitamin D on NK cells is largely unknown. In this study, we demonstrated that CD69(+) activating receptor expression on NK cells was significantly decreased by incubation with 1,25(OH)2 D3 in a dose-dependent manner, while CD158a and CD158b inhibitory receptor expression was upregulated. The degranulation marker CD107a was significantly downregulated on NK cells following incubation with 1,25(OH)2 D3 . NK-cell conjugation with K562 target cells was not affected by 1,25(OH)2 D3 ; however, depolarization of perforin granules in conjugated NK cells was significantly increased. TLR4 expression on NK cells was significantly decreased and TNF-α and IFN-γ production was significantly reduced by 1,25(OH)2 D3 through interference with NF-κB. Our results suggest 1,25(OH)2 D3 has immune regulatory effects on NK cell cytotoxicity, cytokine secretion and degranulation process as well as TLR4 expression. Potential therapeutic application of 1,25(OH)2 D3 for dysregulated NK-cell immunity should be explored in the future.

Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity.

STUDY QUESTION: Do women with recurrent pregnancy losses (RPL) and low vitamin D have increased prevalence of auto- and cellular immune abnormalities when compared with women with RPL who have normal vitamin D, and does vitamin D have any effect on cellular immunity in vitro? SUMMARY ANSWER: A high proportion of women with RPL have vitamin D deficiency and the risk of auto- and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency. WHAT IS KNOWN ALREADY: Vitamin D deficiency in pregnant women is associated with increased risk of obstetrical complications such as pre-eclampsia, bacterial vaginosis associated preterm delivery, gestational diabetes mellitus and small-for-gestational age births. STUDY DESIGN, SIZE, DURATION: A retrospective cross-sectional study of 133 women with RPL who were enrolled in a 2-year period, together with laboratory experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with three or more consecutive spontaneous abortions prior to 20 weeks of gestation who were enrolled at the University clinic. Serum vitamin D level, cellular activity and autoimmune parameters in vivo and in vitro were measured. MAIN RESULTS AND THE ROLE OF CHANCE: Sixty-three out of 133 women (47.4%) had low vitamin D (<30 ng/ml). The prevalence of antiphospholipid antibody (APA) was significantly higher in low vitamin D group (VDlow) (39.7%) than in the normal vitamin D group (VDnl) (22.9%) (P< 0.05) and the adjusted odds ratio (OR) for APA in VDlow was 2.22 with the 95% confidence interval (CI) of 1.0-4.7. The prevalence of antinuclear antigen antibody (VDlow versus VDnl; 23.8% versus 10.0%, OR 2.81, 95% CI 1.1-7.4), anti-ssDNA (19.0% versus 5.7%, OR 3.76, 95% CI 1.1-12.4) and thyroperoxidase antibody (33.3% versus 15.7%, OR 2.68, 95% CI 1.2-6.1) was significantly higher in VDlow than those of VDnl (P < 0.05 each). Peripheral blood CD19(+) B and CD56(+) NK cell levels and NK cytotoxicity at effector to target cell (E:T) ratio of 25:1 were significantly higher in VDlow when compared with those of VDnl (P < 0.05 each). Reduction (%) of NK cytotoxicity (at E:T ratio of 50:1 and 25:1) by IgG (12.5 mg/dl) was significantly lower in VDlow than those of VDnl (P < 0.05, P < 0.01, respectively). There were no differences in Th1/Th2 ratios between VDlow and VDnl. When vitamin D3 was added in NK cytotoxicity assay in vitro, NK cytotoxicity at E:T ratio of 50:1 was significantly suppressed with 10 nMol/L (nM) (11.9 ± 3.3%) and 100 nM (10.9 ± 3.7%) of vitamin D3 when compared with controls (15.3 ± 4.7%) (P < 0.01 each). TNF-α/IL-10 expressing CD3(+)/4(+) cell ratios were significantly decreased with 100 nM of vitamin D3 (31.3 ± 9.4, P < 0.05) when compared with controls (40.4 ± 11.3) in vitro. Additionally, INF-γ/IL-10 expressing CD3(+)/4(+) cell ratio was significantly decreased with 100 nM of vitamin D3 (12.1 ± 4.0, P < 0.05) when compared with controls (14.8 ± 4.6). IFN-γ and TNF-α secretion from NK cells were significantly decreased (P < 0.01 each), and IL-10, IL-1ß, vascular endothelial growth factor and granulocyte colony stimulating factor levels were significantly increased (P < 0.01 each) with vitamin D3 100 nM when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION: The prevalence of vitamin D deficiency in women with RPL in this study is open to a possible type I error since women with vitamin D supplementation were excluded from this study. WIDER IMPLICATIONS OF THE FINDINGS: Assessment of vitamin D level is recommended in women with RPL. Vitamin D supplementation should be explored further as a possible therapeutic option for RPL. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the intramural funding from Department of Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Peripheral blood NK cell cytotoxicities are negatively correlated with CD8(+) T cells in fertile women but not in women with a history of recurrent pregnancy loss.

PROBLEM: We aim to investigate NK cell cytolytic activities and its relationship to other lymphocyte subsets in peripheral blood of women with a history of recurrent pregnancy loss (RPL). METHODS OF STUDY: Women with a history of RPL (n = 48) comprised RPL group, and 15 fertile women served as controls. Lymphocyte subsets such as T (CD3(+)), T helper (CD3(+)/4(+)), cytotoxic T (CD3(+)/8(+)), NK (CD3(-)/56(+)), and peripheral blood NK cell cytolytic activities at three different effector to target cell ratios (E/T ratio, 50:1, 25:1 and 12.5:1) are measured by flow cytometric analysis. RESULTS: Peripheral blood NK cell levels are significantly increased in women with RPL as compared to controls (P = 0.001). NK cell cytolytic activities in RPL group are significantly increased as compared to those of controls at E/T ratio of 50:1 (42.5 ± 16.3 versus 29.9 ± 13.8, P = 0.009), 25:1 (31.6 ± 15.0 versus 19.4 ± 10.1, P = 0.004), and 12.5:1 (20.1 ± 10.9 versus 12.3 ± 7.5, P = 0.011). In RPL group, peripheral blood NK cell levels (%) showed a significant positive correlation with NK cell cytolytic activities at E/T ratio of 50:1 (r = 0.522, P < 0.001), 25:1 (r = 0.588, P < 0.001), and 12.5:1 (r = 0.604, P < 0.001). In controls, CD3(+)/8(+) cells (%) show a negative correlation with NK cell cytolytic activities at E/T ratio of 50:1 (r = -0.566, P = 0.028), 25:1 (r = -0.60., P = 0.017), and 12.5:1 (r = -0.602, P = 0.018). Ratios of T-helper cell to T-cytotoxic cell are positively correlated with NK cell cytolytic activities at E/T ratio of 50:1 (r = 0.601, P = 0.018), 25:1 (r = 0.632, P = 0.012), and 12.5:1 (r = 0.637, P = 0.011). CONCLUSION: NK cell-mediated immunopathology plays a role in RPL. Women with RPL have a disrupted immune regulation between cytotoxic T and NK cells. Failure of immune modulation by CD8(+) T cells may exert NK cell activation and reproductive failures in women with RPL.

Expression of Kisspeptin and its receptor GPR54 in the first trimester trophoblast of women with recurrent pregnancy loss.

PROBLEM: Kisspeptin and its receptor GPR54 play a major role in trophoblast invasion. The expression of kisspeptin and GPR54 in trophoblast and decidua and their relationship with decidual and peripheral blood natural killer (NK) cells are investigated in women with RPL. METHOD OF STUDY: Trophoblast and decidual tissues were collected from 38 RPL women who miscarried a genetically normal fetus and 14 women who had elective abortion. Kisspeptin, GPR54, and decidual NK cells were investigated with immunohistochemistry, and peripheral blood NK cells were analyzed by flow cytometry. RESULTS: Kisspeptin expression in syncytiotrophoblast was significantly decreased in RPL women with normal (<15%) peripheral blood NK cells (npNK) (P=0.021) and high (≥15%) peripheral blood NK cells (hpNK) (P=0.024) as compared to controls. Kisspeptin expression in cytotrophoblast was significantly decreased hpNK group (P=0.009) as compared to controls. GPR54 expressions were not different among study groups and controls. The number of CD56(+) decidual NK cells are significantly higher in hpNK group as compared to npNK group (P=0.041) and showed a correlation with kisspeptin expression in syncytiotrophoblasts (r=0.738, P<0.001). CONCLUSION: Decreased kisspeptin expression in trophoblasts is associated with RPL and kisspeptin may engage the regulation of decidual NK cell infiltration.

Postoperative acute renal failure in patients with gynecologic malignancies: analysis of 10 cases and review of the literature.

OBJECTIVE: Postoperative acute renal failure (PO-ARF) is an important cause of mortality among surgical patients. Although there have been many reports on PO-ARF after cardiac surgery and liver transplantation, less is known about the risk of PO-ARF after gynecologic operations. We aimed to investigate the risk of PO-ARF on gynecologic malignancy operations. METHODS: 1,155 patients' medical charts were reviewed who underwent therapeutic surgery for gynecologic malignancies from January 1, 2005 to December 31, 2007, at the Asan Medical Center, Seoul, Korea. RESULTS: Of these, 10 patients, comprising 0.89% of those who underwent radical hysterectomies and 0.86% of those who underwent debulking operations, were diagnosed with PO-ARF. Their mean age was 61.9+/-10.1 years. Five patients had preoperative risk factors. Mean operating time was 360.8+/-96.2 minutes. Five patients experienced intra-operative hypotension and all patients were given blood transfusions during surgery. Eight patients underwent hemodialysis, with two continuing on dialysis to date. Only two patients fully recovered. CONCLUSION: Patients undergoing surgery for gynecologic malignancies may be at high risk for PO-ARF, because of old age, long operation times, and profuse bleeding. It is necessary to monitor these patients for postoperative renal function and urine output. If a postoperative oliguric state is detected, aggressive volume expansion should be started immediately, followed by hemodialysis.

Pregnancy outcomes using assisted reproductive technology after fertility-preserving therapy in patients with endometrial adenocarcinoma or atypical complex hyperplasia.

OBJECTIVES: To evaluate the outcomes of pregnancy in young women (<40 years old) with early endometrial cancer or atypical complex hyperplasia who were treated by conservative management followed by assisted reproductive technology (ART). MATERIALS AND METHODS: Medical charts of 11 patients treated from January 1997 to October 2007 at Asan Medical Center were retrospectively reviewed. These patients had all been treated with progestin and serial dilatation and curettage as primary fertility-preserving therapies. RESULTS: After pathological remission of disease, 10 patients tried to become pregnant by ART, 4 by in vitro fertilization and embryo transfer, and 6 by controlled ovarian hyperstimulation, with or without intrauterine insemination. Eight women had intrauterine pregnancies, and 6 patients had live births. Patients have been followed up for 9 to 51 months (mean, 21 months) after delivery, with no evidence of tumor recurrence. CONCLUSIONS: Fertility-preserving therapy followed by ART can be a good option in well-selected patients with early endometrial cancer who want to become pregnant.