Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

STUDY QUESTION: Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? SUMMARY ANSWER: EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). WHAT IS KNOWN ALREADY: The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. STUDY DESIGN, SIZE, DURATION: This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. STUDY FUNDING/COMPETING INTEREST(S): This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Plasminogen activator inhibitor-1, factor V, factor II and methylenetetrahydrofolate reductase polymorphisms in women with recurrent miscarriage.

The present study investigated the association between genetic polymorphisms of selected thrombophilic factors with recurrent miscarriage (RM). The genetic polymorphisms for plasminogen activator inhibitor-1 4G/5G (PAI-1), Factor V Leiden (FVL), Factor II G20210A (FII) and methylenetetrahydrofolate reductase MTHFR C677T were determined in 186 RM women and 129 healthy women. In RM women, the frequency of heterozygosity for PAI-1 5G/4G (31%) was significantly higher than in controls (5G/4G: 22%) whereas no difference was found in the case of homozygosity 4G/4G and 5G/5G. The frequencies of genotype G/A for FVL and FII were significantly higher in RM women (FVL, 10%; FII, 8%) than in controls (FVL, 3%; FII, 2%). No difference was found in the case of MTHFR C677T. The polymorphisms of FVL and FII should be screened in RM women, whereas PAI-1 seems to be weakly associated with RM. The role of MTHFR C677T polymorphisms without hyperhomocysteinemia appears negligible.

3D transvaginal ultrasound diagnosis of uterine septa according to different classifications: are there other measurements that correlate to reproductive outcome in small indentation length?

Background: High discrepancy between current classifications was observed in the definition of uterine septa, especially for indentation lengths >5 <10mm. Objectives: To assess the discrepancy between current classifications in the diagnoses of septate uterus and to correlate them with reproductive outcomes; to detect 3D transvaginal ultrasound (TVS) additional measurements, which can better correlate small indentation lengths >5 <10mm to reproductive failures. Materials and Methods: Observational study enrolling 664 women of reproductive age with 3D ultrasound diagnosis of an indentation length ≥3mm. For each patient a detailed reproductive history was taken before performing 3D transvaginal examination. Patients with previous uterine surgery or metroplasty were excluded. Main Outcome Measures: Indentation lengths >5 <10mm showed high discrepancy in the diagnosis of uterine septum between different classifications. For these small indentations additional 3D measurements (indentation angle, septal width and septal length/ fundal myometrial thickness (L/M) ratio) were correlated to infertility and recurrent miscarriage. Results: Among the cohort, 215 patients showed an indentation length >5 <10mm; 136 tried to conceive: 69 (51%) were infertile, 38 (28%) had recurrent miscarriages (≥2) and 5 (4%) had at least one delivery. Recurrent miscarriage significantly correlated to an indentation angle >134°; whereas infertility to an indentation width <32mm and a L/M ratio >75%. Conclusions: Wide discrepancies between different classifications are more evident in indentation lengths >5 <10mm. Additional measurements on 3D coronal section may help to evaluate the risk of infertility or recurrent miscarriage. What is New?: Additional 3D TVS measurements, beyond septal lengths, in particular for small fundal indentation, may help in predicting the risk of developing adverse reproductive outcomes.

Low molecular weight heparin -induced miRNA changes in peripheral blood mononuclear cells in pregnancies with unexplained recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is a clinical condition for which there is a lack of evidenced-based therapies. However, in clinical practice, low molecular weight heparin (LMWH) has been widely used as an empirical therapy since immune effects have been hypothesized in modulating immune tolerance at the fetal-maternal interface. Epigenetic mechanisms are involved in establishing of immune tolerance, at fetal-maternal interface. To investigate potential induced immune-epigenetic changes at maternal periphery level, which could reflect the maternal-fetal interface condition, seems to open up new therapeutical strategies, since microRNAs circulating in maternal plasma and in peripheral blood mononuclear cells (PBMCs) may be specific and sensitive immunological markers/predictors of adverse pregnancy outcomes such as RPL. Our aim in this pilot study is to evaluate potential LMWH effects on genes regulating immunological response key mechanisms related to maternal-fetal tolerance processes, by studying circulating miRNAs in maternal peripheral blood. We tested a panel of selected miRNAs on three groups: 18 healthy pregnant women, 20 pregnant women affected by uRPL, 18 pregnant women affected by uRPL, treated with LMWH. The majority of differentially expressed miRNAs (miR 374a-5p, 19a-3p, 30e-5p, 128-3p, 155-5p and 200c-3p) were found to be modulated by LMWH, which seems to have a positive function in RPL patients, by bringing patients' values back to those comparable to the control ones. Selected microRNA panels would appear to be an effective clinical tool for uRPL diagnosis and management. LMWH-modified miRNA expression levels could be targets for immunotherapy, as LMWH would appear to restore physiological miRNA levels, which are dysregulated in uRPL.

Machine Learning (ML) based-method applied in recurrent pregnancy loss (RPL) patients diagnostic work-up: a potential innovation in common clinical practice.

RPL is a very debated condition, in which many issues concerning definition, etiological factors to investigate or therapies to apply are still controversial. ML could help clinicians to reach an objectiveness in RPL classification and access to care. Our aim was to stratify RPL patients in different risk classes by applying an ML algorithm, through a diagnostic work-up to validate it for the appropriate prognosis and potential therapeutic approach. 734 patients were enrolled and divided into 4 risk classes, according to the numbers of miscarriages. ML method, called Support Vector Machine (SVM), was used to analyze data. Using the whole set of 43 features and the set of the most informative 18 features we obtained comparable results: respectively 81.86 ± 0.35% and 81.71 ± 0.37% Unbalanced Accuracy. Applying the same method, introducing the only features recommended by ESHRE, a correct classification was obtained only in 58.52 ± 0.58%. ML approach could provide a Support Decision System tool to stratify RPL patients and address them objectively to the proper clinical management.

Pregnancy-promoting actions of HCG in human myometrium and fetal membranes.

Human chorionic gonadotropin (HCG) plays a major role in early human development through a series of well recognized pregnancy-promoting actions that are exerted in the first trimester, including maternal recognition of pregnancy, enhancement of embryo implantation and survival, stimulation of trophoblast growth and differentiation, and prolongation of the functional life of the corpus luteum. Recent research indicates that HCG can exert significant pregnancy-promoting actions also in the remainder of pregnancy through its effect on the myometrium and on fetal membranes. In the myometrium, HCG promotes the inhibition of smooth muscle cell contractility through several mechanisms, including inhibition of gap junction formation, reduction of intracellular calcium concentration, increase in the expression of progesterone receptor, and an increase in the expression of phosphodiesterase 5 (PDE5), an enzyme controlling the intracellular levels of cGMP. This effect appears to be specific for PDE5 since it has not been found for other hormones potentially involved in pregnancy such as estrogen, progesterone and thyroid hormone. In fetal membranes, HCG can modulate expression of the inducible isoform of nitric oxide synthase (iNOS), as well as specific immunoregulatory cytokines such as the high mobility group box 1 (HMGB1) protein. This accumulating evidence suggests that HCG has a wide spread pregnancy-promoting actions that are exerted in various reproductive and gestational tissues.

Uterine blood flow indices, antinuclear autoantibodies and unexplained recurrent miscarriage.

OBJECTIVE: To study the correlation between 2D and 3D uterine flow indexes and the presence or the absence of antinuclear antibodies (ANA) in women with unexplained recurrent miscarriage (uRM). METHODS: Fifty-two subjects (26 uRM and 26 control women) underwent 2D Doppler measurement of pulsatility index and resistance index of the uterine arteries in both the follicular and midluteal phase of the cycle. Additionally, 3D ultrasonography determination of vascularisation index, flow index, and vascularisation flow index was carried out with the aid of the VOCAL technique. Serum assay for the presence of ANA was performed in all women. RESULTS: Pulsatility index of ANA+ uRM women was higher than that of ANA- uRM women and control ANA+ and ANAwomen, both in the follicular and in the midluteal phase of the cycle. Vascularisation index in ANA- uRM women was significantly higher than that in ANA+ control women. Flow index in uRM ANA+ women was significantly lower than that of each of the other groups. CONCLUSION: ANA might be involved in uRM by determining an impairment in uterine blood flow hemodynamic, particularly in uterine blood flow intensity and uterine artery impedance.

Urocortin stimulates placental adrenocorticotropin and prostaglandin release and myometrial contractility in vitro.

Urocortin is a new member of the CRF family. Multiple biological effects for urocortin have been shown in rats and in some in vitro models, showing a modulatory role in hormonal and behavioral functions. Human placenta expresses urocortin, but no information is available on the possible local biological actions. The aim of the present study was to evaluate the effect of urocortin on placental ACTH and prostaglandin (PG) secretion, as well as on myometrial contractility. Various in vitro models were used. For investigating the effect of urocortin on ACTH release, primary cultures of human trophoblast cells were used. Culture media, collected before and after 3 h exposure to different doses of urocortin and ACTH, were measured by RIA. Trophoblast tissue explants were incubated for 24 h in the presence of increasing doses of urocortin, and prostaglandin E2 (PGE2) levels were measured by RIA. Strips of myometrial tissue were incubated in an organ bath and connected to an isometric smooth-muscle transducer in the presence of urocortin, with or without prostaglandin F2alpha (PGF2a). In all these experiments, the effect of astressin (a CRF receptor antagonist) on urocortin-induced actions and the effect of equimolar doses of CRF were evaluated. A dose-related increase of trophoblast ACTH or PGE2 was induced by urocortin, whereas astressin inhibited urocortin-stimulated ACTH or PGE2 release. Equimolar doses of CRF showed a similar effect on both ACTH and PGE2. Urocortin increased PGF2alpha-induced myometrial contractility, and this effect was completely abolished by the addition of astressin. The present study showed that human urocortin stimulates placental secretion of ACTH and PGE2, and modulates myometrial contractility, suggesting a role for this peptide in placental and intrauterine CRF pathways.

Interleukin-3 stimulates prostaglandin E2 release by human fetal membranes at term gestation.

The effect of interleukin-3 (IL-3) on prostaglandin E2 (PGE2) release by human fetal membranes at term gestation was evaluated. It was found that interleukin-3 stimulates in vitro prostaglandin E2 release by these gestational tissues, suggesting that interleukin-3 might be involved in the regulation of arachidonic acid metabolism in the fetal membranes.

Interleukin-2 in human amniotic fluid during pregnancy and parturition: implications for prostaglandin E2 release by fetal membranes.

The potential role of interleukin 2 (IL-2) in human pregnancy was investigated by evaluating the following. (1) The presence and concentrations of IL-2 in amniotic fluid (AF) in 24 women at 16-18 weeks' gestation (Group 1) and in 27 women at term pregnancy, either before the onset of labor (Group 2, n = 10) or during spontaneous active labor (Group 3, n = 17). (2) The production of IL-2 by fetal membranes at term gestation (n = 7). (3) The release of prostaglandin E2 (PGE2) by the above tissues after stimulation with IL-2 (n = 10) or phytohemagglutinin (PHA) (n = 8). Immunoreactive IL-2 was detected only in AF samples obtained from women of Groups 1 and 3; the higher concentration was found in Group 1 samples; IL-2 was not detected in AF samples from women of Group 2. Tissues did not release IL-2. Both IL-2 and PHA exerted a significant stimulatory effect on PGE2 release by tissues. IL-2 stimulated PGE2 release by chorion tissue, but not by amnion tissue. The following conclusions can be drawn: (a) AF IL-2 might play a role in the maternal-fetal immune relationship during early pregnancy and, perhaps, during labor; (b) fetal membranes would not seem to represent a source of AF IL-2 in the absence of labor; (3) IL-2 might influence arachidonic acid metabolism through the cyclooxygenase pathway in the chorion tissue.

Hormonal regulation of cytokine release by human fetal membranes at term gestation: effects of oxytocin, hydrocortisone and progesterone on tumour necrosis factor-alpha and transforming growth factor-beta 1 output.

Inflammatory cytokines can play an important role in the biomolecular processes leading to labour by regulating prostaglandin production in intrauterine tissues. In the setting of intrauterine infection, an increased production of these cytokines by placenta, decidua and fetal membranes occurs and is responsible for the onset and maintenance of preterm labour. However, the factors involved in the control of cytokine release by these tissues in normal pregnancy at term are still largely unknown. We investigated the possibility that the synthesis and release of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) by human fetal membranes at term gestation is regulated by several hormones potentially involved either in the maintenance of pregnancy or in the parturitional process. In the present study, the effects of hydrocortisone, progesterone and oxytocin on TNF-alpha and TGF-beta1 release by explants of fetal membranes at term gestation were evaluated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the effect of the above hormones on mRNA expression; TNF-alpha and TGF-beta1 release in culture medium was quantitifed by ELISA assays. Results show that both tissue mRNA expression for TNF-alpha and TNF-alpha release in culture medium were significantly increased by oxytocin, but not by hydrocortisone and progesterone. On the contrary, all the hormones tested increased both tissue TGF-beta1 mRNA expression and release in culture medium. These findings suggest that TNF-alpha and TGF-beta1 production by human fetal membranes in uncomplicated pregnancy at term is selectively modulated by oxytocin, hydrocortisone and progesterone.

Oxytocin in human intrauterine tissues at parturition.

The recent detection of oxytocin (OT) mRNA in human gestational tissues suggests that OT may be locally synthesized and released to act on the uterus as a local mediator in the mechanism of parturition. In order to investigate this possibility the OT immunoreactive (I.R.) content was examined directly in placental decidua and amniochorial membranes after term and preterm delivery and in their culture media at term gestation. I.R.OT concentrations were also measured in maternal, retroplacental and umbilical plasma as well as in amniotic fluid in the presence or the absence of labour. Low I.R.OT concentrations (below 15 fmol g-1 wet tissue) were found in both amniochorial membranes and placental decidua. Moreover, whereas in amniochorion they were higher (P < 0.05) after preterm than term spontaneous parturition, in decidua they were higher (P < 0.05) after term than preterm vaginal delivery. Detectable amounts (below 15 fmol g-1 wet tissue per h) of I.R.OT were also found in culture media from explants of the above tissues. Among all the examined maternal and fetal fluids a rise in I.R.OT content at parturition was detected only in the amniotic liquor (P < 0.05). These findings suggest that I.R.OT concentrations in intrauterine tissues are very low; however, considering that OT is the most potent endogenous uterotonic agent, OT as such concentrations might play a paracrine function in the biochemical events leading to human parturition. Therefore, a role for amniotic OT in parturition can not be excluded.

Nitric oxide in human fetal membranes at term gestation: effect on prostaglandin E2 release.

OBJECTIVES: To examine the in vitro release of nitric oxide (NO) by human fetal membranes at term gestation and to investigate whether NO could stimulate prostaglandin E2 (PGE2) release by these tissues. STUDY DESIGN: Explants of fetal membranes (n = 17) were incubated either in the presence of sodium nitroprusside (NP), or L-Arginine (L-Arg), or bacterial lipopolysaccharide (LPS), or in the absence of the above substances (controls). NO and PGE2 concentrations in culture medium were assayed by the Griess reaction and radioimmunoassay, respectively. RESULTS: Fetal membranes spontaneously released NO in culture medium; incubation with NP increased the production of both NO and PGE2. L-Arg and LPS enhanced PGE2 output by tissues but did not influence NO production. CONCLUSIONS: An NO-generating activity might be present in human fetal membranes. NO stimulates PGE2 release by these tissues.