Stefano Raffaele Giannubilo

Placental thrombomodulin expression in recurrent miscarriage

BackgroundEarly pregnancy loss can be associated with trophoblast insufficiency and coagulation defects. Thrombomodulin is an endothelial-associated anticoagulant protein involved in the control of hemostasis and inflammation at the vascular beds and its also a cofactor of the protein C anticoagulant pathway.DiscussionWe evaluate the Thrombomodulin expression in placental tissue from spontaneous recurrent miscarriage and voluntary abortion as controls. Thrombomodulin mRNA was determined using real-time quantitative polymerase chain reaction. Reduced expression levels of thrombomodulin were found in recurrent miscarriage group compared to controls (1.82-fold of reduction), that corresponds to a reduction of 45% (from control group Delta CT) of thrombomodulin expression in spontaneous miscarriage group respect the control groups.SummaryWe cannot state at present the exact meaning of a reduced expression of Thrombomodulin in placental tissue. Further studies are needed to elucidate the biological pathway of this important factor in the physiopathology of the trophoblast and in reproductive biology.

Uterine Fibroids: Pathogenesis and Interactions with Endometrium and Endomyometrial Junction

Uterine leiomyomas (fibroids or myomas) are benign tumors of uterus and clinically apparent in a large part of reproductive aged women. Clinically, they present with a variety of symptoms: excessive menstrual bleeding, dysmenorrhoea and intermenstrual bleeding, chronic pelvic pain, and pressure symptoms such as a sensation of bloatedness, increased urinary frequency, and bowel disturbance. In addition, they may compromise reproductive functions, possibly contributing to subfertility, early pregnancy loss, and later pregnancy complications. Despite the prevalence of this condition, myoma research is underfunded compared to other nonmalignant diseases. To date, several pathogenetic factors such as genetics, microRNA, steroids, growth factors, cytokines, chemokines, and extracellular matrix components have been implicated in the development and growth of leiomyoma. This paper summarizes the available literature regarding the ultimate relative knowledge on pathogenesis of uterine fibroids and their interactions with endometrium and subendometrial myometrium.

Transabdominal amnioinfusion in preterm premature rupture of membranes: a randomised controlled trial

Objective  To evaluate the role of transabdominal amnioinfusion in improving the perinatal outcomes of pregnancies complicated by preterm premature rupture of membranes (pPROM).

Amniotic vascular endothelial growth factor (VEGF) and nitric oxide (NO) in women with subsequent preeclampsia.

OBJECTIVE To assess whether amniotic fluid concentrations of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in early pregnancy correlate to subsequent preeclampsia. STUDY DESIGN We performed a retrospective study to assess VEGF and NO on the second trimester amniotic fluid of 15 healthy women, and 15 women who subsequently developed preeclampsia. RESULTS In women with subsequent preeclampsia, both VEGF (213.19+/-78.42 pg/ml) and NO concentrations (4.31+/-1.02 micromol/mg creatinine) were significantly lower than healthy controls (VEGF 255.05+/-88.66 pg/ml; NO 5.02+/-1.57 microg/mg creatinine; P<0.05). CONCLUSIONS Our findings suggest that reduced VEGF may be responsible, at least in part, for the impaired vascular development which occurs in preeclampsia. Low concentrations of VEGF and NO in the second trimester may represent an impaired stimulus to vascular formation and endothelial regulation that induce placental disease and preeclampsia.

Number and size of uterine fibroids and obstetric outcomes

Abstract Objective: Estimating the impact of sonographically identified multiple or large (≥5 cm in diameter) fibroids on obstetric outcomes. Methods: Retrospective cohort study of 219 women with uterine fibroids (identified on a routine second-trimester ultrasound survey over a 3-year period, 2010–2012) and their age-matched controls. Inclusion criteria were singleton pregnancy, delivery at >24 weeks of gestation and no pathological conditions (chronic hypertension, gestational diabetes or pre-existing diabetes mellitus, uterine anomalies or fetal malformations). Results: Compared to women with no fibroids, women with multiple fibroids (n = 34) had a significantly higher rate of preterm birth (29.4% versus 5%, p < 0.001), cesarean section (73.5% versus 37%, p < 0.001) and breech presentation (11.8% versus 2.7%, p = 0.04). Women with large fibroids (n = 48) had a higher rate of preterm birth (16.7% versus 5%, p = 0.01) and pPROM (10.4% versus 0.5%, p < 0.001). By multivariate analysis, only multiple fibroids and previous preterm birth showed an independent significant association with preterm birth (OR = 7.37, 95% CI 2.50–21.68 and OR = 13.01, 95% CI 3.56–47.52, respectively). Conclusions: Women with uterine fibroids are at an increased risk of obstetric complications. In particular, multiple rather than large fibroids are associated with a significantly increased risk of preterm birth and cesarean delivery while large fibroids are associated with a higher risk of pPROM. No correlation with IUGR, placenta previa or placental abruption was found.

Expression Patterns of Two Serine Protease HtrA1 Forms in Human Placentas Complicated by Preeclampsia with and without Intrauterine Growth Restriction

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function

CONTEXT Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. OBJECTIVE The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. DESIGN This was a laboratory study. SETTING Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. PATIENTS The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. INTERVENTIONS Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. RESULTS We found that activin-A and myostatin significantly reduce cell proliferation in primary myometrial cells but not in leiomyoma cells as measured by a CyQUANT cell proliferation assay kit. Reduced expression of proliferating cell nuclear antigen and Ki-67 were also observed in myometrial cells in response to activin-A and myostatin treatment. Activin-A also significantly increased mRNA expression of fibronectin, collagen1A1, and versican in primary leiomyoma cells. Finally, we found that activin-A and myostatin activate Smad-2/3 signaling but do not affect ERK or p38 signaling in both myometrial and leiomyoma cells. CONCLUSIONS This study results suggest that activin-A and myostatin can exert antiproliferative and/or fibrotic effects on these cell types via Smad-2/3 signaling.

HLA-G and pregnancy adverse outcomes

There is growing evidence that pregnancy complications such as preeclampsia, recurrent pregnancy loss (RPL), IUGR, and premature birth could be associated with abnormal immunologic interactions at the fetal-maternal interface. The restricted expression of HLA-G to the subpopulation of trophoblast cells which invade the uterus has generated much interest. The alternative splicing of HLA-G primary transcript, gives origin to seven isoforms, including both membrane-bound forms (HLA-G1, G2, G3, G4) and soluble forms (sHLA-G: sHLA-G5, G6, G7). sHLA-G consists predominantly of sHLA-G1 after its shedding by metalloproteinases, and secreted sHLA-G5 representing the quantitatively dominating and full-length isoforms. HLA-G expression and HLA-G genetic variations in both the mother and the embryo/fetus may be important for pregnancy outcome. It is also intuitively apparent that a gene with putative immunosuppressive and immunotolerant potential might be functional in both the mother and the embryo/fetus/placenta. Reduced or aberrant HLA-G expression seems to be associated with certain complications of pregnancy, among which preeclampsia and possibly the risk of miscarriage, and that this may be further linked to HLA-G polymorphisms. Most of the studies aimed at assessing the role of HLA-G in pregnant diseases have considered only the maternal genotype and ignored the contribution of the fetus. In this regard, the mother, placenta and the fetus form a synthesis. Therefore, studies on placental diseases should address HLA-G expression and genetic variations also to the fetus/placenta.

Preeclampsia: No longer solely a pregnancy disease

Preeclampsia, the leading cause of maternal and perinatal morbidity and mortality, has been recently considered not only a pregnancy disease but also a risk factor for developing diseases later in life. Preeclampsia is becoming a disease of interest to internists and not just obstetricians. Women who have had preeclampsia seem to be at higher risk of premature death, mortality from ischemic heart disease, cardiovascular diseases including ischemic heart disease and hypertension, fatal and non-fatal stroke, venous thromboembolism, renal failure, type 2 diabetes mellitus, hypothyroidism, and cognitive defects, although they appear surprisingly protected from cancer. Furthermore, having had preeclampsia is a problem not only for the mothers future health, but it also affects the offsprings adult health. Children born from preeclamptic pregnancies are more prone to hypertension, insulin resistance and diabetes mellitus, neurological problems, stroke, and mental disorders along their life. Whether preeclampsia is a risk factor for disease later in life or it creates long-term organ damage is an intriguing question. This review analyzes recent epidemiological evidence of the long-term outcomes of preeclampsia and the background mechanisms of this phenomenon. Understanding the etiological background may provide guidance for the prevention and follow-up of women who experience preeclampsia.

The involvement of inflammatory cytokines in the pathogenesis of recurrent miscarriage.

OBJECTIVE To investigate the inflammatory cytokine expression pattern in trophoblastic tissue from women with unexplained recurrent miscarriage (RM). STUDY DESIGN Trophoblasts were obtained during uterine evacuation from 11 women with RM and from 20 healthy pregnant women undergoing elective termination of pregnancy, who served as controls. The array was performed using GEArray Q Series Human Inflammatory Cytokines & Receptors Gene Array HS-015 membranes. Data were confirmed by quantitative real-time PCR. The Mann-Whitney U test was performed for statistical analysis. RESULTS Microarray analysis identified three genes that were differentially expressed between RM patients and controls. We observed significant downregulation of Transforming Growth Factor beta 3 (TGF-β3) and Interleukin 25 (IL-25) (5-fold reduction and 2.5-fold reduction, respectively) and significant upregulation of CD-25, also known as Interleukin 2 receptor alpha (IL-2RA) (7-fold increase) in women with RM compared with controls. The median ΔC(t) of TGF-β3 was 8.2 (interquartile range, 7.67-8.9) in RM patients vs. 5.85 (interquartile range, 5.3-6.09) in controls; the median ΔC(t) of IL-25 was 5.18 (interquartile range, 4.46-5.76) in RM patients vs. 3.85 (interquartile range, 3.6-4.51) in controls, and the median ΔC(t) of CD-25 was 9.62 (interquartile range, 7.81-12.42) in RM patients vs. 12.44 (interquartile range, 11.02-13.86) in controls. DISCUSSION Our results suggest that the immunological and inflammatory regulation mechanisms of the placental environment play a key role in recurrent miscarriage. The observed trophoblast cytokine expression pattern at the maternal-fetal interface confirms the immunotrophic theory, as demonstrated by a switch from a T-helper-1 (Th1) profile to a T-helper-2 (Th2) profile in women who experience recurrent miscarriages.