Evaluation of etiology and pregnancy outcome in recurrent miscarriage patients

The purpose of this study was to evaluate etiology and pregnancy outcome of recurrent miscarriage women. The enrolled patients (280) were evaluated for Triiodothyronine, Thyroxine, Thyroid stimulating hormone, prolactin, chromosomal analysis, Haemoglobin A1C, blood sugar, Magnetic resonance imaging, 3D-ultrasound, auto-antibodies profile (antiphospholipid antibodies, anticardiolipin antibodies, lupus anticoagulant, antinuclear antibodies, anti-thyroid antibodies and β2 glycoprotein1), torch profile (Toxoplasmo gondii, rubella, cytomegalo virus and herpes simplex virus), blood vitamin D3 levels, psychological factors, Body mass index and thrombotic factors (protein S and C deficiency, Prothrombin G20210A mutation, anti-thrombin III, Factor V Leiden and Methylenetetrahydrofolate reductase mutation), uterosalpingography (hysteronsalpingography) and hysteroscopy. The therapeutic regimens either singly or combined were employed for the treatment of recurrent miscarriage patients on the basis of etiology (single or multiple) and include intravenous immunoglobulin, low molecular weight heparin, low dose aspirin, levothyroxine, progesterone, folic acid, human chorionic gonadotrophin, vitamin D3, psychotherapy, genetic counselling. However, patients with idiopathic recurrent miscarriage were treated with progesterone supplementation, anticoagulation and/or immune modulatory agents. The incidence of primary recurrent miscarriage was highest and most of the women experienced recurrent miscarriage during first trimester. Endocrinological disorders (39%) were found as the major pathological factor for recurrent miscarriage. Other factors include uterine abnormalities (5.7%), vitamin D3 deficiency (3.5%), psychological factors (3.2%) infection (3.6%), autoimmune abnormalities (1.8%) and protein S deficiency (1.8%). However, 40% cases were idiopathic. The overall live birth rate achieved after the management of recurrent miscarriage patients was 75.7%. Enocrinopathy was the major cause of recurrent miscarriage. The overall live birth rate achieved was 75.7% with highest pregnancy outcome in secondary recurrent miscarriage patients after the management.     

Novel mechanism of miRNA‐365‐regulated trophoblast apoptosis in recurrent miscarriage

Clinical pregnancies increasingly end in recurrent miscarriage (RM) during the first trimester, with genetic factors shouldering the main responsibility. MicroRNAs (miRNAs) regulate gene expression in a wide array of important biological processes. We examined the potential role of dysregulated miRNAs in RM pathogenesis and trophoblast development as an approach to elucidate the molecular mechanism behind RM. miRNA profiles from clinical specimens of RM and induced abortion (IA) were compared, and several miRNAs were found to be aberrantly expressed in RM samples. Among the miRNAs, miR‐365 was significantly differentially expressed in RM decidual tissues. Furthermore, our results demonstrate that miR‐365 functions as an upstream regulator of MDM2/p53 expression, cell cycle progression and apoptosis in trophoblasts. Bioinformatic prediction and experimental validation assays identified SGK1 as a direct target of miR‐365; consistently, its protein levels were low in decidual tissues. Additionally, functional studies revealed that SGK1 silencing elicits cell cycle arrest and apoptosis in trophoblasts and that SGK1 overexpression attenuates the effects of miR‐365 on apoptosis and MDM2/p53 expression. Collectively, our data provide evidence that the up‐regulation of miR‐365 may contribute to RM by decreasing SGK1 expression, which suggests its potential utility as a prognostic biomarker and therapeutic target for RM.     

INO80 participates in the pathogenesis of recurrent miscarriage by epigenetically regulating trophoblast migration and invasion

The INO80 complex, a SWI/SNF family chromatin remodeler, has regulatory effects on ESC self-renewal, somatic cell reprogramming and blastocyst development. However, the role of INO80 in regulating trophoblast cells and recurrent miscarriage (RM) remains elusive. To investigate the in vivo effects of Ino80 in embryo development, we disrupted Ino80 in C57 mice, which resulted in embryonic lethality. Silencing of Ino80 led to decreased survival capacity, migration and invasion of trophoblasts. Furthermore, RNA high-throughput sequencing (RNA-seq) revealed that Ino80 silencing closely resembled the gene expression changes in RM tissues. To investigate the mechanisms for these results, RNA-seq combined with high-throughput sequencing (ChIP-seq) was used in trophoblast cells, and it showed that Ino80 physically occupies promoter regions to affect the expression of invasion-associated genes. Last, Western blotting analyses and immunofluorescence staining revealed that the content of INO80 was reduced in RM patients compared to in healthy controls. This study indicates that INO80 has a specific regulatory effect on the viability, migration and invasion of trophoblast cells. Combined with its regulation of the expression of invasion-associated genes, it has been proposed that epigenetic regulation plays an important role in the occurrence of RM, potentially informing RM therapeutic strategies.     

The role of dydrogesterone in recurrent (habitual) abortion

The published evidence regarding the administration of dydrogesterone in the treatment of habitual abortion is summarised in this review. Habitual abortion is defined as the loss of three or more consecutive pregnancies without known maternal or foetal pathology. The immunology of early pregnancy seems to determine the rejection or non-rejection of the allogenic embryo. When peripheral mononuclear cells from recurrent aborters are incubated with progesterone or dydrogesterone in vitro, T-helper (Th)2 cytokines such as interleukin (IL)-4 and IL-6 markedly increase whereas the Th1 cytokine interferon-γ decreases. Additionally, both progesterone and dydrogesterone are thought to inhibit the activity of natural killer cells at the foeto-maternal interface in humans. Progesterone-induced blocking factor (PIBF) mediates the immunological effects of progesterone and dydrogesterone in pregnancy. It affects various phases of the maternal immune response involving both the cellular and humoral immune system, exerts anti-abortive effects and inhibits the release of arachidonic acid. It also favours the production of so-called asymmetric, pregnancy-protecting antibodies. In rodents, blockade of this factor results in the termination of pregnancy and in women considerably lower levels are found in those with threatened abortion or pre-term labour. In order to draw final conclusions as to the usefulness of dydrogesterone in women with a history of recurrent miscarriage, further controlled, blinded, randomised clinical trials are needed.     

The miR-27a-3p/USP25 axis participates in the pathogenesis of recurrent miscarriage by inhibiting trophoblast migration and invasion

Insufficient invasion ability of trophoblasts might be associated with the development of recurrent miscarriage (RM). Ubiquitin-specific protease 25 (USP25) can regulate the processes of invasion and migration in different types of cancer cells. However, the effect of USP25 on trophoblasts and its roles in the development of RM are unknown. In this study, we first analyzed the USP25 expression in placental villous tissues from RM patients, and then assessed the roles of USP25 in epithelial-to-mesenchymal transition (EMT), invasion and migration of trophoblasts. Furthermore, bioinformatics prediction and luciferase reporter assay were used to explore the mechanism of microRNA on USP25 expression, and regulation of USP25 expression in trophoblasts was assessed following transfection with microRNA mimics or inhibitor. The results showed that the expression of USP25 in the placental villous tissues was downregulated in RM patients. Knockdown of USP25 suppressed the EMT process, the invasion and migration capability of trophoblast cells, while overexpression of USP25 exhibited opposite results. Mechanistically, miR-27a-3p could regulate USP25 expression by binding to the 3′-untranslated region of USP25 in trophoblasts. Quantitative real-time polymerase chain reaction results found the expression of miR-27a-3p were negatively related to USP25 in RM patients. MiR-27a-3p mimics inhibited but miR-27a-3p inhibitor enhanced the migration and invasion capability of trophoblasts. Furthermore, sh-USP25 counteracted the promotion of invasion and migration mediated by the miR-27a-3p inhibitor. Taken together, these data indicate that USP25 downregulation by miR-27a-3p contributes to the EMT process, thereby inhibiting the migration and invasion of trophoblast cells, and these findings might provide potential biomarkers for RM.     

Intravenous immunoglobulin G treatment increases live birth rate in women with recurrent miscarriage and modulates regulatory and exhausted regulatory T cells frequency and function

Exhausted T cells and regulatory T (Treg) cells have been recently proposed to be new risk factors for recurrent miscarriage (RM). Intravenous immunoglobulin G (IVIG) treatment reported to modulate various immune cells. In this study, the effects of IVIG on the frequency and function of exhausted T cells, exhausted Tregs, and Treg cells, as well as pregnancy outcome in women with unexplained RM (URM), were investigated. Ninety-four pregnant women with RM were enrolled. At the time of positive pregnancy, blood samples were drawn. Forty-four patients with URM were included as IVIG receiving treated group and received 400 mg/kg of IVIG and the rest fifty patients were considered as a control group and received no IVIG administration. IVIG was given intravenously every 4 weeks during 32 weeks of gestation. Blood samples of patients were collected after the latest administration. Exhausted T cells, exhausted Tregs, and Treg cells were evaluated pre- and posttreatment in both groups. IVIG induced a significant decrease in the frequency of exhausted Tregs population and function as well as a significant increase in Treg cells population, however, IVIG failed to affect population and the function of exhausted T cells. Pregnancy outcome was successful in IVIG treated women (86.3%) and were significantly different (P = 0.0006) in compared with the untreated URM subjects (42%). Therefore, employing of IVIG increases Treg cells and diminishes exhausted Tregs responses in RM patients with cellular immune anomalies throughout the pregnancy. Immunemodulatory effects of IVIG are probably associated with successful pregnancy outcome.     

Analysis of CCR5 and CX3CR1 gene polymorphisms in association with unexplained recurrent miscarriages among north Indian women

Context

Recurrent miscarriage (RM), defined as three or more consecutive losses before the 20th week of gestation, affects 0.5–2% of pregnant women. In over 80% of cases, RM remains unexplained after investigations, suggesting the involvement of genetic factors.

Objectives

The present study investigates the common polymorphisms of chemokine receptors CCR5 (NG_012637.1:g.5303A>G) and CX₃CR1 (NG_016362.1:g.21065C>T, Thr280Met and NG_016362.1:g.20971G>A, Val249Ile) and their association with recurrent miscarriages (RM) among north Indian women.

Participants and Methods

In a retrospective case-control study 200 well characterized patients with unexplained RM and 300 controls were genotyped for three polymorphic markers of CCR5 and CX₃CR1 by restriction digestion of PCR amplified fragments.

Results

Alleles and genotypes of CX₃CR1 Val249Ile revealed statistically significant associations with RM cases when compared with the controls. The homozygous variant genotype Ile/Ile was found to be significantly higher among patients (p = 0.0002) when compared with the homozygous wild type Val/Val genotype. The haplotype of CX₃CR1 that carried major alleles of Thr280Met and Val249Ile (T-V) showed statistically significant protective association (p < 0.0001, OR = 0.41, 95% CI = 0.31–0.54). The haplotype A-T-V (all wild type alleles) revealed a statistically significant protective association (p < 0.0001, OR = 0.41, 95% CI = 0.34–0.62), whereas the haplotypes G-T-I, A-T-I and A-M-V modified the risk of RM 1.9-fold, 5.5-fold and 5.1-fold respectively.

Conclusions

A common polymorphism of CX₃CR1 gene, Val240Ile is associated with the risk of RM in north Indian women. Risk of RM may also be modified by the presence of haplotypes T-I, M-V, G-T-I, A-T-I and A-M-V.     

Potential innate immunity-related markers of endometrial receptivity and recurrent implantation failure (RIF)

The successful implantation of the embryo into a receptive endometrium is essential for the establishment of a viable pregnancy while recurrent implantation failure (RIF) is a real challenge in assisted reproduction. The maternal innate immune system, specifically the Toll-like receptors (TLRs), are involved in maintaining immunity in the female reproductive tract (FRT) required for fertility. In this study, we aimed to investigate the importance of innate immunity-related gene expression in the regulation of human fertility and as a prediction of potential outcome of in vitro fertilization - embryo transfer (IVF-ET), thus, we assessed the gene expression levels of TLR signalling molecules using quantitative real-time PCR between endometrial biopsies of healthy fertile women, and the patients experiencing RIF. Interestingly, our results showed that, TRIB2 and TLR9 genes were differentially expressed between the endometrial biopsies of healthy women and those with RIF. However, comparing expression levels of same genes between pre-receptive and receptive healthy endometrial biopsies showed different genes (ICAM1, NFKBIA, VCAM1, LIF, VEGFB, TLR5) had significantly altered expression, suggesting their involvement in endometrial receptivity. Thus, further investigations will enable us to better understand the role of these genes in the biology of FRT and as a possible target for the improvement of infertility treatments and/or development of non-hormonal contraception.     

Natural killer cell receptor expression in patients with severe and recurrent Herpes simplex virus-1 (HSV-1) infections

Herpes simplex virus-1 (HSV-1) is an important human pathogen which in a minority of people causes severe infections. In immunocompetent hosts the infection is self limiting. However, a small minority of people have frequent attacks. As NK cells have been implicated in host protection against HSV-1, the aim of this study was to compare NK cell receptor expression in healthy controls and in patients suffering from recurrent HSV-1 reactivations using monoclonal antibodies against NK cell receptors and 3 colour flow cytometry. Eighteen patients were recruited into the study and the results were compared to a control group. The results obtained showed that overall there was no statistical difference between patient and control groups in the expression of the NK cell receptors. There were however, individuals in the patient group (in particular, two members of one family) with significantly reduced level of activating receptors compared to the control group.     

Molecular investigation of association between common IL-6 polymorphism with cytomegalovirus (CMV) infection and recurrent miscarriage in Iranian women

Molecular Biology Reports - Recurrent pregnancy loss (RPL) is described as two or more spontaneous abortions. To date, scientists in various fields of knowledge, such as genetics, endocrinology,...     

Assessment of DNA damage in relation to heavy metal induced oxidative stress in females with recurrent pregnancy loss (RPL)

Pregnancy termination consecutively for three or more times during the first trimester is termed as Recurrent pregnancy loss (RPL). In addition to the abnormal karyotype, heavy metal induced oxidative damage may contribute as prominent etiological factor in pregnancy termination. Oxidative stress is considered crucial in etiology underlying RPL with altered antioxidant status and subsequent DNA damage. The current case controlled study investigated Total antioxidant capacity (TAC), DNA damage (8OHdG) and heavy metals in RPL group (n = 30) and the women with successful pregnancies and no cases of miscarriage as control group (30 women). Heavy metals -Antimony (Sb) and Arsenic (As) were measured by Inductively Coupled Plasma Mass spectrophotometry (ICP-MS). There was significant decrease in levels of TAC in RPL group compared to healthy pregnant women (P < 0.05). On contrary, elevated levels of As and Sb were observed in RPL group with subsequent increase in the levels of 8OHdG (P < 0.001); indicating extensive DNA damage in these patients. Furthermore, increased levels of As and Sb in RPL group were positively correlated with 8OHdG and negatively with total antioxidant capacity. The outcome of the study provides clear insight of the role of metal induced oxidative stress that plays a vital role in the pathophysiology underlying RPL.     

A Comparison of the Structures of the Alpha:Beta and Alpha:Gamma Dimers of Mouse Salivary Androgen-Binding Protein (ABP) and Their Differential Steroid Bindin

Mouse salivary androgen-binding protein (ABP) isa family of dimeric proteins that may play a pheromonalrole in Mus musculus. The protein dimer consists of acommon alpha subunit disulfide-bonded to avariable (beta or gamma) subunit. Here wereport N-terminal sequences of the beta and gammasubunits, showing that they are very similar to eachother while being quite different from the alphasubunit.We demonstrate differential androgen binding bythe two dimers. Both bind dihydrotestosterone to aboutthe same extent but the alpha:beta dimer bindssignificantly more testosterone than the alpha:gammadimer. We discuss the possible significance ofthis diversity of androgen binding with respect to thepossibility that androgen binding is related to aputative pheromonal role for the protein.     

Mutation Analysis of Inhibitory Guanine Nucleotide Binding Protein Alpha (GNAI) Loci in Young and Familial Pituitary Adenomas

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gα_i) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1 _, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gα_i proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gα_i proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.     

The Genes for Mouse Salivary Androgen-Binding Protein (Abp) Subunits Alpha and Gamma Are Located on Chromosome 7

We demonstrate that the previously described gene Androgen binding protein (Abp; Dlouhy and Karn, 1984) codes for the Alpha subunit of ABP and rename the locus Androgen binding protein alpha (Abpa). A study of recombinant inbred strains demonstrates that Abpa is located on chromosome 7 near Glucose phosphate isomerase-1 (Gpi-1). Biochemical and genetic evidence indicates the existence of another ABP subunit, Gamma, and its locus, Androgen binding protein gamma (Abpg), that is closely linked to Abpa. Although no polymorphism has yet been found for the previously described Beta subunit of ABP (Dlouhy and Karn, 1983; 1984), we suggest that it represents a third locus. Androgen binding protein beta (Abpb). ABP subunits appear to dimerize randomly and a model is presented demonstrating the origin of six ABP dimers in the salivas of AbpaaAbpga/AbpabAbpgb heterozygous mice. The results of cell-free translation studies in which the pre-ABP subunits are identified specifically by immunoprecipitation with anti-ABP antibody supports the idea that independent mRNAs code for the Alpha, Beta and Gamma subunits.     

Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies)

OBJECTIVE: To determine whether treatment with low dose aspirin and heparin leads to a higher rate of live births than that achieved with low dose aspirin alone in women with a history of recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies), lupus anticoagulant, and cardiolipin antibodies (or anticardiolipin antibodies). DESIGN: Randomised controlled trial. SETTING: Specialist clinic for recurrent miscarriages. SUBJECTS: 90 women (median age 33 (range 22-43)) with a history of recurrent miscarriage (median number 4 (range 3-15)) and persistently positive results for phospholipid antibodies. INTERVENTION: Either low dose aspirin (75 mg daily) or low dose aspirin and 5000 U of unfractionated heparin subcutaneously 12 hourly. All women started treatment with low dose aspirin when they had a positive urine pregnancy test. Women were randomly allocated an intervention when fetal heart activity was seen on ultrasonography. Treatment was stopped at the time of miscarriage or at 34 weeks'' gestation. MAIN OUTCOME MEASURES: Rate of live births with the two treatments. RESULTS: There was no significant difference in the two groups in age or the number and gestation of previous miscarriages. The rate of live births with low dose aspirin and heparin was 71% (32/45 pregnancies) and 42% (19/45 pregnancies) with low dose aspirin alone (odds ratio 3.37 (95% confidence interval 1.40 to 8.10)). More than 90% of miscarriages occurred in the first trimester. There was no difference in outcome between the two treatments in pregnancies that advanced beyond 13 weeks'' gestation. Twelve of the 51 successful pregnancies (24%) were delivered before 37 weeks'' gestation. Women randomly allocated aspirin and heparin had a median decrease in lumbar spine bone density of 5.4% (range -8.6% to 1.7%). CONCLUSION: Treatment with aspirin and heparin leads to a significantly higher rate of live births in women with a history of recurrent miscarriage associated with phospholipid antibodies than that achieved with aspirin alone.     

Alpha-hemoglobin-stabilizing protein (AHSP) in hemolysis,elevated liver enzyme,and low platelet (HELLP) syndrome,intrauterine growth restriction (IUGR) and fetal death

Objective Alpha hemoglobin-stabilizing protein (AHSP) inhibits the production of reactive oxygen species in various cells, including erythrocytes. Reduced AHSP can mean reduced protection from stressors. Our objective was to investigate whether AHSP is involved in the response to stress in pregnancy. Study design Placentas were collected from normal term pregnancies (n = 10) and pregnancies complicated by HELLP (n = 10), intrauterine growth restriction (IUGR; n = 10) or fetal death (IUFD; n = 6). AHSP messenger RNA (mRNA) and protein were determined using real time quantitative polymerase chain reaction (PCR) and Western blot, respectively. All statistical analyses were performed by using the GraphPad Prism Software. Differences were considered significant at p < 0.05. Results Placental AHSP mRNA level in HELLP (4.16E10⁻⁴ ± 1.77) and IUFD (4.19E10⁻⁴ ± 3.37) were significantly decreased compared with controls (28.47E10⁻⁴ ± 14.86; p < 0.01), whereas levels in the IUGR group (7.55E10⁻⁴ ± 6.4) showed a trend toward being lower but the difference did not reach statistical significance. Western blot analysis results indicate a no significant increase of ASHP protein in the HELLP syndrome group and a significant decrease in the IUFD group compared with controls. There was no significant difference between the IUGR and control groups. Conclusion ASHP mRNA expression in the placenta is decreased in complicated pregnancies, and it may be involved in the pathogenic mechanisms leading to the adverse pregnancy outcome. This paper was presented as a poster at the 27th Annual Meeting of the Society for Maternal Fetal Medicine; San Francisco, CA, USA, February 5–10, 2007. Monica Emanuelli and Davide Sartini contributed equally to this paper.     

Placental retention in a bonobo (Pan paniscus)

Background  This case report describes the first placental retention in an 11-year-old female bonobo ( Pan paniscus ) following the delivery of a healthy infant.
Methods  After unsuccessful medical treatment with oxytocin, the placenta was manually extracted.
Results and conclusions  Both the dam and infant survived.