Unexplained Recurrent Spontaneous Miscarriage Research Articles

Decreased β-catenin Expression in First Trimester Villi and Decidua of Patients with Recurrent Spontaneous Miscarriage

Accumulating evidence suggests that Wnt/β-catenin pathways play an important role in the physiological processes of endometrial development and differentiation, preimplantation embryo development, and blastocyst implantation. The objective of the current study was to signify the association between Wnt/β-catenin signalling pathway and recurrent spontaneous miscarriage and examine the expression of β-catenin first-trimester villi and decidua in women with spontaneous miscarriage. A comparative cross-sectional study was conducted in Saladin General Hospital from the 1st of January to the 1st of October 2021. A convenient sample of 80 pregnant women was enrolled in the current study and consisted of group A (40 pregnant women who were presented with first or second miscarriage) and group B (40 pregnant women who were presented with recurrent spontaneous miscarriage). There was a significant association between decreased β-catenin and recurrent miscarriage as 77.5% of the women who presented with recurrent spontaneous miscarriage had decreased levels of β-catenin, while only 32.5% of those with first or second miscarriage had decreased levels of β-catenin. The results of the current study implied that β-catenin is a valuable biomarker for unexplained recurrent spontaneous miscarriage. The women who presented with recurrent spontaneous miscarriage had a decreased expression of β-catenin .

WISP2/IGF1 promotes the survival of DSCs and impairs the cytotoxicity of decidual NK cells

The survival and development of a semi-allogeneic fetus during pregnancy require the involvement of decidual stromal cells (DSCs), a series of cytokines and immune cells. Insulin-like growth factor 1 (IGF-1) is a low molecular weight peptide hormone with similar metabolic activity and structural characteristics of proinsulin, which exerts its biological effects by binding with its receptor. Emerging evidence has shown that IGF-1 is expressed at the maternal-fetal interface, but its special role in establishment and maintenance of pregnancy is largely unknown. Here, we found that the expression of IGF-1 in the decidua was significantly higher than that in the endometrium. Additionally, decidua from women with normal pregnancy had high levels of IGF-1 compared with that from women with unexplained recurrent spontaneous miscarriage. Estrogen and progesterone led to the increase of IGF-1 in DSCs through up-regulating the expression of WISP2. Recombinant IGF-1 or DSCs-derived IGF-1 increased the survival, reduced the apoptosis of DSCs, and downregulated the cytotoxicity of decidual NK cells (dNK) through interaction with IGF-1R. These data suggest that estrogen and progesterone stimulate the growth of DSCs and impair the cytotoxicity of dNK possibly by the WISP2/IGF-1 signaling pathway.

Aspirin Enhances the Protective Effect of Progesterone on Trophoblast Cell from Oxidative Stress and Apoptosis

Objective: Clinically, low-dose aspirin and progesterone are frequently used to prevent pregnancy loss. We investigated the effect of these drugs on the biological behavior of human extravillous trophoblasts in vitro. Methods: HTR-8/SVneo cells were cultured in vitro and treated with different concentrations of aspirin and progesterone. The proliferation, invasion, and apoptosis of HTR-8/SVneo cells were assessed using a cell counting Kit-8 assay, Matrigel Transwell assay, and Hoechst staining, respectively. Reverse transcriptase polymerase chain reaction was used to verify the expression of related genes. Reactive oxygen species (ROS) levels were detected using the 2,7-dichlorofluorescin diacetate assay. Results: Low-dose aspirin alone, progesterone alone, or aspirin plus progesterone upregulated the proliferation and invasion and decreased the apoptosis of HTR-8/SVneo cells. Moreover, the expression of marker of proliferation Ki-67 (MKI67), matrix metalloproteinases 2 (MMP2), and MMP9 was increased. In addition, low-dose aspirin plus progesterone exerted stronger anti-apoptosis effects than low-dose aspirin and progesterone alone. Interestingly, aspirin upregulated the expression of progesterone receptor (PGR). Treatment with hydrogen peroxide (H2O2) promoted ROS production in HTR-8/SVneo cells; however, low-dose aspirin plus progesterone significantly restricted H2O2-mediated ROS production and apoptosis in HTR-8/SVneo cells. Conclusions: These data suggest that low-dose aspirin and progesterone promote proliferation and invasion and cooperatively reduce oxidative stress and apoptosis in trophoblasts in vitro. These results may provide an experimental basis for the combined application of aspirin and progesterone to prevent unexplained recurrent spontaneous miscarriage, especially in patients with trophoblast dysfunction.

Effects of Progesterone, Dydrogesterone and Estrogen on the Production of Th1/Th2/Th17 Cytokines by Lymphocytes from Women with Recurrent Spontaneous Miscarriage

Anti-inflammatory Th2 cytokines have been shown to be associated with healthy, successful pregnancy while pro-inflammatory Th1 and Th17 cytokines are associated with pregnancy loss due to recurrent spontaneous miscarriage. This nexus between unexplained recurrent spontaneous miscarriage (uRSM) and maternal inflammatory has led to the possibility of using pregnancy-related hormones to modify the maternal cytokine bias in a manner that is conducive to successful pregnancy. We investigated the ability of progesterone, dydrogesterone and estrogen to modulate cytokine production by peripheral blood lymphocytes from women undergoing uRSM. Peripheral blood mononuclear cells (PBMC) from females with uRSM were stimulated in vitro with phytohemagglutinin (PHA) in the presence and absence of progesterone or dydrogesterone or 17β-estradiol. Culture supernatants were assayed for IFN-α, TNF-γ, IL-2, IL-6, IL-10, IL-13, IL-17A, and IL-23 by ELISA. Progesterone and dydrogesterone significantly down-regulated the secretion of the Th1 cytokines IFN-α and TNF-γ, and the Th17 cytokine IL-17A, and IL-23. Additionally, the secretion of the Th2 cytokine IL-6 was up-regulated. Estrogen, on the other hand, decreased the production of IFN-α and IL-2, increased the production of IL-6 but did not affect IL-17A and IL-23 secretion. Progestogens and estrogen can decrease the production of some Th1/Th17 inflammatory cytokines secreted by lymphocytes from uRSM and upregulate the production of anti-inflammatory cytokines. These data support the notion that progestogens can be used for altering maternal cytokine profiles to manage pregnancy complications.

Low anti-Müllerian hormone concentration is associated with increased risk of embryonic aneuploidy in women of advanced age.

Does an association exist between serum anti-Müllerian hormone (AMH) level, the marker of biological ovarian age, and embryonic aneuploidy risk in recurrent spontaneous miscarriage (RSM) patients of reproductive age? This retrospective study included a total of 422 IVF cycles of 394 unexplained RSM patients undergoing preimplantation genetic testing for aneuploidy (PGT-A), enrolled from January 2014 to December 2016. Subjects were divided into three groups according to the 25th (1.50 ng/ml) and 75th (5.60 ng/ml) percentiles of AMH level (Group 1: low AMH <1.50 ng/ml [N = 107], Group 2: normal AMH 1.50- < 5.60 ng/ml [N = 210] and Group 3: high AMH ≥ 5.60 ng/ml [N = 105]). There was a significant difference in embryonic aneuploid rate between AMH groups (66.7% versus 42.9% versus 50.0%, Groups 1 to 3, respectively, P = 0.006). It was significantly higher in the low AMH group (Group 1) compared with that in the normal AMH group (Group 2, P1vs2 = 0.002) and high AMH group (Group 3, P1vs3 = 0.015). After age stratification, embryonic aneuploidy rate was still significantly different among AMH groups with a similar trend in women ≥35 years old (68.2% versus 54.4% versus 51.0%, P = 0.038, P1vs2 = 0.025, P1vs3 = 0.035), but not in young subjects. These findings indicate that low AMH level was associated with increased risk of embryo aneuploidy only in women of advanced age. Maternal diminished ovarian reserve along with oocyte ageing may contribute to impaired chromosomal competence of the embryo.

Th17 cells and related cytokines in unexplained recurrent spontaneous miscarriage at the implantation window

Unexplained recurrent spontaneous abortion (RSA) might be caused by the mother's immunological rejection of the fetus. In this cross-sectional study, the percentage of T helper 17 (Th17), T regulatory (Treg) cells and their cytokines as the main players of immunomodulation in peripheral blood lymphocytes during the luteal phase of 20 women with unexplained RSA were compared with 20 normal non-pregnant women. The percentage of Treg cells in the former was significantly lower compared with controls. The percentage of Th17 cells in the former was higher than controls. Expression of IL-23, IL-17, IL-6 cytokines in the former was significantly higher than controls, but the higher expression of IL-21 was not significant. The gene expression of TGF-β and FoxP3 in the former was lower than controls. Significant positive correlations were found between the percentage of Th17 cells with IL-23, IL-6 and IL-17 and between expression of IL-23 and IL-6 and IL-17. IL-6 gene expression showed a significant positive correlation with IL-17. Therefore, imbalance of Th17–Treg cells and the consequent changes in cytokine expression might be implicated in the pathogenesis of unexplained RSA and may provide new insight into the immunoregulatory events at the maternal–fetal interface.

Increased Dickkopf-1 expression in patients with unexplained recurrent spontaneous miscarriage

Wnt pathways play an important role in pre-implantation embryo development, blastocyst implantation, and post-implantation uterine decidualisation. However, little is known about the potential role that Wnt signaling plays in patients with unexplained recurrent spontaneous miscarriage (URSM), and no single biomarker with a high predictive value of maternally caused URSM has been identified. We aim to study the molecular mechanisms by which the Wnt pathway controls the progression of early pregnancy by investigating the expression of Dickkopf-1 (DKK1), one of the Wnt agonists, in URSM patients. Plasma and fresh decidual tissues samples were collected from 59 subjects (29 patients with URSM and 30 patients with normal, early pregnancy). Time-resolved immunofluorometric assay system and quantitative real-time RT-PCR were used to determine the serum levels of DKK1 and DKK1 mRNA in the deciduas, respectively. Western blot and immunohistochemistry were used to measure DKK1 protein levels in the deciduas. Serum DKK1 levels were significantly higher in URSM patients compared to the control group (P < 0·001); the expression of DKK1 mRNA and protein in URSM patients were higher relative to healthy controls (P = 0·013). Glandular epithelium from decidual tissues demonstrated cytoplasmic signals for DKK1 in URSM patients, and DKK1 did not stain in healthy controls. Furthermore, serum DKK1 levels significantly correlated with those in the decidual tissues. Our study suggests that DKK1 may be a valuable biomarker of URSM; it can be reliably and conveniently detected in serum, thus obviating the need for decidual tissue analysis.

Increased expression of Toll-like receptor 3 in decidual natural killer cells of patients with unexplained recurrent spontaneous miscarriage

To investigate whether the expression of Toll-like receptor 3 (TLR3) in decidual natural killer (dNK) cells plays a role in the pathogenesis of unexplained recurrent spontaneous miscarriage (URSM). NK cells were isolated from human decidual tissue through magnetic cell sorting and analyzed for phenotypes through flow cytometry. TLR3 expression in dNK cells was detected by quantitative reverse-transcriptase polymerase chain reaction and western blotting. The cytotoxicity of dNK was detected using a lactate dehydrogenase release test. The CD56(+) dNK percentages varied between 85% and 96%. The expression of TLR3 mRNA and protein in dNK cells of URSM patients was statistically significantly higher than that in control women (p=0.005 and p=0.01, respectively). Meanwhile, the mean cytotoxicity percentage of freshly isolated dNK cells from the patients was significantly higher than that from normal controls (31.24 ± 7.46% vs. 16.20 ± 6.03%; p=0.03). The expression of TLR3 in dNK cells is abnormally higher in URSM patients, which indicates that TLR3 expression may be critical to the activation of dNK cells infiltrating the maternal-fetal interface and account for the loss of immune tolerance.

Decidual CD4 +CD25 +CD127 dim/− regulatory T cells in patients with unexplained recurrent spontaneous miscarriage

Objectives To investigate the frequency and function of CD4 +CD25 +CD127 dim/− regulatory T (Treg) cells in decidua of patients with unexplained recurrent spontaneous miscarriage (URSM). Study design The decidual lymphocytes from patients who experienced URSM and normal pregnant women (controls) were collected by Ficoll density gradient centrifugation. CD4 +CD25 +CD127 dim/− Treg cells were isolated by magnetic cell sorting. The proportion of Treg cells and IL-10, TGF-β in Treg cells were determined by flow cytometry. Inhibitory effects of Treg cells on effecter T cells were detected with or without the presentation of anti-IL-10 antibodies and anti-TGF-β antibodies. Results The frequency of CD4 +CD25 +CD127 dim/− Treg cells was decreased in URSM decidua compared to controls (2.09% ± 0.86% vs. 2.97% ± 1.19%, p = 0.005), and the expression of IL-10 and TGF-β in Treg cells was lower in the URSM group than in the control group ( p = 0.04 and p = 0.01, respectively). Furthermore, the suppressive effect of CD4 +CD25 +CD127 dim/− Treg cells on the proliferation of effector T cells was decreased in URSM decidua ( p < 0.05). Suppression was mediated predominantly through IL-10 and TGF-β in decidua. Conclusions Decreased frequency and immunosuppressive capacity of CD4 +CD25 +CD127 dim/− Treg cells was found in URSM decidua. Treg cells inhibit proliferation of effector T cells mainly via IL-10 and TGF-β in URSM decidua.

Serum levels of TNF-α and antioxidant enzymes and placental TNF-α expression in unexplained recurrent spontaneous miscarriage

It has been proposed that many factors have been implicated in the pathogenesis of unexplained recurrent spontaneous miscarriage (URSM). The objective of this study was to evaluate the levels of some antioxidants, tumor necrosis factor-alpha (TNF-alpha), luteinizing (LH) and follicle-stimulating hormone (FSH) in URSM. Serum levels of superoxide dismutase (SOD), catalase (CAT), LH, FSH as well as TNF-alpha in serum and the expression of TNF-alpha positive cells in placental tissues, were assayed in women suffering from unexplained first trimester miscarriage. Two groups were included, the first was represented by 16 women with URSM (number of abortions: 3-5) and the second one included 24 women with URSM (number of abortions > 5). The control groups included 20 women within their first trimester of pregnancy and 20 non pregnant healthy females within their follicular phase. The obtained results showed a highly significant decrease in serum levels of SOD and CAT, in the URSM groups compared to control groups (p < 0.05 for each comparison). Higher serum levels of TNF-alpha were detected in URSM groups compared to control groups (p < 0.05 for each comparison). A significant increase in serum levels of LH was encountered between URSM groups compared to control groups; on the other hand the mean levels of FSH expressed no significant changes among URSM groups as compared to first trimester pregnancies control group. A positive correlation was noticed between serum levels of TNF-alpha and the levels of LH (p < 0.05). We conclude that antioxidant enzymes (CAT, SOD), TNF-alpha, LH and FSH may play a major role in the pathogenesis of URSM. Much more work is required before the mechanisms, which lead to RSM, can be fully understood.

Progesterone-induced blocking factor (PIBF) modulates cytokine production by lymphocytes from women with recurrent miscarriage or preterm delivery

Spontaneous miscarriage and preterm delivery are common complications of pregnancy. Pro-inflammatory cytokines have been shown to be associated with recurrent spontaneous miscarriage (RSM) and preterm delivery (PTD) and these have led to exploration of ways to downregulate pro-inflammatory cytokines and/or to upregulate anti-inflammatory cytokines. Progesterone-induced blocking factor (PIBF) is a molecule with inhibitory effects on cell-mediated immune reactions. We have ascertained the effects of PIBF on secretion of selected type 1 and type 2 cytokines by peripheral blood mononuclear cells from healthy non-pregnant women, women undergoing normal pregnancy, women with unexplained RSM and women with PTD. Peripheral blood mononuclear cells from 30 women with a history of unexplained RSM, 18 women undergoing PTD, 11 women with normal pregnancy and 13 non-pregnant healthy women were stimulated with a mitogen in the absence and presence of PIBF after which the levels of cytokines released into culture supernatants were determined by ELISA. Production of the type 2 cytokines IL-4, IL-6 and IL-10 by lymphocytes from the RSM and PTD groups and of IL-4 and IL-10 by lymphocytes from healthy pregnant women was significantly increased upon exposure to PIBF, while the levels of type 1 cytokines were not affected. Ratios of type 1:type 2 cytokines were decreased, suggesting a shift towards Th2 bias. PIBF did not affect cytokine production by lymphocytes from non-pregnant women. Thus, PIBF acts on lymphocytes in pregnancy to induce a type 1 to type 2 cytokine shift by upregulating the production of type 2 cytokines.

Biochemical role of intravaginal sildenafil citrate as a novel antiabortive agent in unexplained recurrent spontaneous miscarriage: first clinical study of four case reports from Egypt

Many factors have been implicated in the pathogenesis of unexplained recurrent spontaneous miscarriage (URSM). The objective of this preliminary clinical study was to verify whether intravaginal sildenafil citrate tablets (25 mg intravaginally, 4 times/day for 24 days) are effective as a novel treatment of threatened miscarriage in patients with a history of URSM (n=4). We also evaluated changes in the concentrations of some antioxidants, the percent expression of cells positive for tumor necrosis factor-alpha (TNF-alpha), CD56+T-cells [CD3+CD56+ natural killer cells (NKT cells) (cluster differentiation 3, 56 NKT cells)], which express one of the NK cell markers, and uterine artery pulsatility index (PI). We compared these results with healthy women in their first trimester of pregnancy (FTP n=4). Sildenafil improved the measured antioxidants concentrations [total antioxidant capacity (TAC), glutathione S-transferase (GSH-S-T) and catalase (CAT), superoxide dismutase (SOD)], and improved oxidative stress by reducing malondialdehyde (MDA) and nitric oxide (NO) in the four patients under investigation. The improvement in antioxidants was close to concentrations seen in women in their FTP. The four patients had progressed to their third trimester of pregnancy using this novel application. The percentage of [CD3+CD56+ NKT cells] and TNF-alpha in peripheral blood was higher in URSM patients compared to women in their FTP. However, concentrations were greatly reduced and improved after sildenafil administration. We demonstrated that intravaginal sildenafil citrate tablets can reduce vasoconstriction by increasing blood flow through relaxation of uterine arteries as indicated from measured PI in URSM patients. We conclude that intravaginal sildenafil citrate tablets used as suppositories might be a novel, interesting, safe antiabortive option in the treatment of threatened miscarriage in patients with a history of URSM. We are the first to report this novel clinical application in four case reports in Egypt.

Modulation of cytokine production by dydrogesterone in lymphocytes from women with recurrent miscarriage

To examine the effects of dydrogesterone on the production of Th1 and Th2 cytokines by lymphocytes from women undergoing unexplained recurrent spontaneous miscarriage (RSM). Controlled prospective, clinical study conducted in a maternity hospital and a university-based immunology laboratory. Faculty of Medicine, Kuwait University and Kuwait Maternity Hospital. Thirty women with unexplained RSM. Peripheral blood mononuclear cells (PBMC) from women with unexplained RSM were isolated from venous blood by density gradient sedimentation and stimulated with phytohaemagglutinin (PHA). Culture supernatants assayed for interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-4, IL-6 and IL-10 by ELISA. Levels of the progesterone-induced blocking factor (PIBF) were also measured. Cytokine production in the presence and absence of progesterone and dydrogesterone. Dydrogesterone significantly inhibited the production of the Th1 cytokines IFN-gamma (P= 0.0001) and TNF-alpha (P= 0.005) and induced an increase in the levels of the Th2 cytokines IL-4 (P= 0.03) and IL-6 (P= 0.017) resulting in a substantial shift in the ratio of Th1/Th2 cytokines. The effect of dydrogesterone was blocked by the addition of the progesterone-receptor antagonist mifepristone, indicating that dydrogesterone was acting via the progesterone receptor. Dydrogesterone induced the production of PIBF. Dydrogesterone inhibits the production of the Th1 cytokines IFN-gamma and TNF-alpha from lymphocytes and up-regulates the production of the Th2 cytokines IL-4 and IL-6, inducing a Th1 to Th2 cytokine shift.

WITHDRAWN: Human chorionic gonadotrophin for recurrent miscarriage.

There may be an association between recurrent miscarriage and abnormal hormone function in the follicular phase. Human chorionic gonadotrophin may play a role in preventing miscarriages. The objective of this review was to assess the effects of human chorionic gonadotrophin administration during early pregnancy on the risk of miscarriage in women with a history of recurrent miscarriage. The Cochrane Pregnancy and Childbirth Group trials register was searched. Date of last search: 9 January 1998. Randomised trials of human chorionic gonadotrophin compared with placebo or no treatment in women who have had two or more miscarriages. Eligibility and trial quality were assessed by one reviewer. Four trials involving 180 women were included. The trials were of variable quality. Human chorionic gonadotrophin was associated with a reduced risk of miscarriage for women with a history of recurrent miscarriage (odds ratio 0.26, 95% confidence interval 0.14 to 0.52). This result should be interpreted cautiously because the apparent effect is greatly influenced by the two methodologically weaker studies. There is not enough evidence to evaluate the use of human chorionic gonadotrophin during pregnancy in order to prevent miscarriage in women with a history of unexplained recurrent spontaneous miscarriage.

Human chorionic gonadotrophin for recurrent miscarriage.

There may be an association between recurrent miscarriage and abnormal hormone function in the follicular phase. Human chorionic gonadotrophin may play a role in preventing miscarriages. The objective of this review was to assess the effects of human chorionic gonadotrophin administration during early pregnancy on the risk of miscarriage in women with a history of recurrent miscarriage. The Cochrane Pregnancy and Childbirth Group trials register was searched. Date of last search: 9 January 1998. Randomised trials of human chorionic gonadotrophin compared with placebo or no treatment in women who have had two or more miscarriages. Eligibility and trial quality were assessed by one reviewer. Four trials involving 180 women were included. The trials were of variable quality. Human chorionic gonadotrophin was associated with a reduced risk of miscarriage for women with a history of recurrent miscarriage (odds ratio 0.26, 95% confidence interval 0.14 to 0.52). This result should be interpreted cautiously because the apparent effect is greatly influenced by the two methodologically weaker studies. There is not enough evidence to evaluate the use of human chorionic gonadotrophin during pregnancy in order to prevent miscarriage in women with a history of unexplained recurrent spontaneous miscarriage.