Enrrico Bloise

Growth factors and myometrium: biological effects in uterine fibroid and possible clinical implications

BACKGROUND Growth factors are proteins secreted by a number of cell types that are capable of modulating cellular growth, proliferation and cellular differentiation. It is well accepted that uterine cellular events such as proliferation and differentiation are regulated by sex steroids and their actions in target tissues are mediated by local production of growth factors acting through paracrine and/or autocrine mechanisms. Myometrial mass is ultimately modified in pregnancy as well as in tumour conditions such as leiomyoma and leiomyosarcoma. Leiomyomas, also known as fibroids, are benign tumours of the uterus, considered to be one of the most frequent causes of infertility in reproductive years in women. METHODS For this review, we searched the database MEDLINE and Google Scholar for articles with content related to growth factors acting on myometrium; the findings are hereby reviewed and discussed. RESULTS Different growth factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heparin-binding EGF (HB-EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and TGF-β perform actions in myometrium and in leiomyomas. In addition to these growth factors, activin and myostatin have been recently identified in myometrium and leiomyoma. CONCLUSIONS Growth factors play an important role in the mechanisms involved in myometrial patho-physiology.

Impaired Placental Nutrient Transport in Mice Generated by in Vitro Fertilization

More than 4.5 million children have been conceived by in vitro fertilization (IVF). Interestingly, singleton IVF offspring born at term have an increased incidence of low birth weight. The mechanism responsible for the lower birth weight is unknown, but alterations in placental function are possible. Hence, the goal of our study was to examine placental growth and function in mice generated in vivo or in vitro. To assess placental function, blastocysts were generated by IVF or produced by natural mating (control group); both IVF and control blastocysts were transferred to pseudopregnant recipients. Placental weights did not differ at embryonic d 15.5 (E15.5) but were increased at E18.5 in the IVF group (25.4%, P < 0.001) compared with control. Proliferation was increased in IVF placentae, whereas overall placental gross morphology and apoptosis were not affected. Both fetal weights (16.4% lower at E15.5 and 8.8% lower at E18.5, P < 0.05) and fetal to placental ratios were lower (P < 0.001) in the IVF compared with the control group at both time points, whereas birth weights did not differ. At E18.5, the mRNA for selected glucose, system A amino acid transporters, and imprinted genes were down-regulated in IVF placentae. GLUT3 protein level was decreased in the IVF group (P < 0.05). Importantly, intrajugular injections of (14)C-methyl-D-glucose or (14)C-MeAIB tracers (n = 6 litters per group) showed that placental transport of glucose and amino acids were 24.8% (not significant) and 58.1% (P < 0.05) lower in the IVF group. Fetal accumulation of glucose was not different, but amino acid accumulation was significantly (36 %) lower in IVF fetuses (P < 0.05). We conclude that IVF alters both fetal and placental growth and, importantly, decreases placental transport efficiency in mice conceived by IVF.

Urocortin increases IL-4 and IL-10 secretion and reverses LPS-induced TNF-alpha release from human trophoblast primary cells.

Problem  As urocortin (Ucn) is a placental peptide belonging to the corticotrophin‐releasing hormone (CRH) family that modulates immune function in other biological models, this study evaluated Ucn effects on cytokines secretion from cultured human trophoblast cells.

Behavioral sensitization produced by a single administration of apomorphine: Implications for the role of Pavlovian conditioning in the mediation of context-specific sensitization

The present study examined the minimal number of exposures to the D1/D2 agonist apomorphine capable of producing behavioral sensitization. Rats received one (experiment 1) or two administrations on two successive days (experiment 2) of apomorphine (0.5 and 2.0 mg/kg) paired or unpaired to an open-field environment. After 2 days of drug withdrawal, the rats received a challenge injection with the same dose of apomorphine (sensitization test) and locomotion, rearing and sniffing were measured. The results of the first experiment showed that locomotor sensitization occurred after a single acute exposure to apomorphine and that 0.5 and 2.0 mg/kg treatments were equally effective. This sensitization effect was context-specific and was limited to locomotion. The second experiment revealed a differential dose effect on the sensitization test. Two treatments with 2.0 mg/kg potentiated locomotor sensitization as compared with a single treatment but two treatments with 0.5 mg/kg did not increase the sensitization effect more than the single 0.5 mg/kg treatment. This result indicates an interaction between drug dose and frequency of drug treatment for the induction of apomorphine locomotor sensitization. In that the sensitization effects are considered to be a core contributor to psychostimulant addiction, the present findings are of importance to understanding addiction because they indicate that sensitization processes can be initiated with a single drug experience and amplified with exposure to higher drug dosage levels.

Activin-A and Myostatin Response and Steroid Regulation in Human Myometrium: Disruption of Their Signalling in Uterine Fibroid

CONTEXT Investigation of activin-A (A) and myostatin (M) in human myometrium (HM) and leiomyoma (HL) will explain their involvement in human myometrial pathophysiology. OBJECTIVE We aimed to investigate A and M response and steroid regulation in HM. We also evaluated A and M expression and response in HL. DESIGN Tissues were analyzed and cultured. PATIENTS Patients included fertile (in proliferative phase) and menopausal women undergoing hysterectomy. INTERVENTIONS HM explant cultures were treated with A and M (for Smad-7 mRNA quantification) or estrogen and progesterone (for A and M mRNA quantification). A and M expression levels were also evaluated in menopausal (physiological absence of steroids) HM specimens. A and M and their receptors were evaluated in HL (n = 8, diameter 5-8 cm) compared with their matched HM. HL explants cultures were treated with A and M (for Smad7 mRNA quantification), and, to explain the absence of response, the levels of follistatin, follistatin-related gene (FLRG), and Cripto were evaluated. RESULTS A and M increased Smad7 expression in HM explants. A and M mRNAs were both reduced after estradiol treatment, unchanged after progesterone treatment, but were higher in menopausal than fertile (in proliferative phase) specimens. A, M, and FLRG were expressed at higher levels in HL compared with adjacent HM, whereas the receptors, follistatin, and Smad7 mRNAs resulted unchanged. Cripto mRNA was expressed only in HL. CONCLUSIONS A and M act on human HM and are regulated by steroids. In HL there is an increase of A, M, FLRG, and Cripto expression.

Comparative intrauterine development and placental function of ART concepti: implications for human reproductive medicine and animal breeding

BACKGROUND The number of children conceived using assisted reproductive technologies (ART) has reached >5 million worldwide and continues to increase. Although the great majority of ART children are healthy, many reports suggest a forthcoming risk of metabolic complications, which is further supported by the Developmental Origins of Health and Disease hypothesis of suboptimal embryo/fetal conditions predisposing adult cardiometabolic pathologies. Accumulating evidence suggests that fetal and placental growth kinetics are important features predicting post-natal health, but the relationship between ART and intrauterine growth has not been systematically reviewed. METHODS Relevant studies describing fetoplacental intrauterine phenotypes of concepti generated by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) in the mouse, bovine and human were comprehensively researched using PubMed and Google Scholar. Intrauterine growth plots were created from tabular formatted data available in selected reports. RESULTS ART pregnancies display minor but noticeable alterations in fetal and placental growth curves across mammalian species. In all species, there is evidence of fetal growth restriction in the earlier stages of pregnancy, followed by significant increases in placental size and accelerated fetal growth toward the end of gestation. However, there is a species-specific effect of ART on birthweights, that additionally vary in a culture condition-, strain-, and/or stage at transfer-specific manner. We discuss the potential mechanisms that underlie these changes, and how they are affected by specific components of ART procedures. CONCLUSIONS ART may promote measurable alterations to intrauterine growth trajectory and placental function. Key findings include evidence that birthweight is not a reliable marker of fetal stress, and that increases in embryo manipulation result in more deviant fetal growth curves. Because growth kinetics in early life are particularly relevant to adult metabolic physiology, we advise more rigorous assessment of fetal growth and placental function in human ART pregnancies, as well as continued follow-up of ART offspring throughout post-natal life. Finally, strategies to minimize embryo manipulations should be adopted whenever possible.

Effect of Substrate Stiffness on Early Mouse Embryo Development

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.

ORIGINAL ARTICLE: Urocortin Increases IL-4 and IL-10 Secretion and Reverses LPS-induced TNF-α Release from Human Trophoblast Primary Cells

Problem  As urocortin (Ucn) is a placental peptide belonging to the corticotrophin‐releasing hormone (CRH) family that modulates immune function in other biological models, this study evaluated Ucn effects on cytokines secretion from cultured human trophoblast cells.

Heat-killed Lactobacillus rhamnosus GG Modulates Urocortin and Cytokine Release in Primary Trophoblast Cells

A number of studies are showing that probiotic treatment induces an anti-inflammatory state. Intrauterine infection can lead to preterm delivery by modulating immune function and efforts to prevent this condition are ongoing nowadays. Lactobacillus rhamnosus GG (LGG) is a probiotic known to ameliorate inflammation by increasing local anti-inflammatory mediators in urinary and gastrointestinal tracts. The present study then analyzed the effect of heat-killed LGG over β-hCG, progesterone, interleukins (IL) 4 and 10, tumor necrosis factor-α (TNF-α), corticotropin releasing hormone (CRH) and urocortin (Ucn) release by primary trophoblast cells. Normal human term placentas (n = 6) were collected and purified trophoblast cells were incubated in the presence of LGG, lipopolysaccharide (LPS) or either LGG + LPS during 3 h, after which the target substances were quantified by ELISA and real-time PCR. LGG did not affect β-hCG, progesterone, or CRH secretion. Conversely, LGG increased IL-4 protein and mRNA expression (P < 0.05) while IL-10 and Ucn secretion were increased in a dose dependent manner and the highest dose of LGG increased significantly IL-10 mRNA (P < 0.05). LGG did not alter TNF-α, while LPS exposure increased TNF-α protein (P < 0.001) and mRNA expression (P < 0.01). Conversely, LGG treatment reversed LPS-induced TNF-α release at both protein (P < 0.01) and mRNA levels (P < 0.05) in a dose dependent fashion. In conclusion, LGG stimulates IL-4, IL-10 and Ucn expression and reverses LPS-induced TNF-α release from trophoblast cells, with no change in β-hCG or progesterone release, suggesting that this probiotic may play a role as an immunomodulatory agent in human placenta without altering basic trophoblast functions.

Polymorphisms of FAS and FAS ligand genes in preeclamptic women

OBJECTIVES This study investigated the influence that Fas and Fas ligand gene polymorphisms might have on preeclampsia. The pathogenesis of preeclampsia is still enigmatic and several studies have proposed that it may, in part, be determined by genetic susceptibility. Therefore, the identification of a gene polymorphism associated with an increased risk of preeclampsia might well represent a useful tool in the identification of at risk pregnant women enabling the setup of preventive therapy. Apoptosis has also been implied in the pathogenesis of preeclampsia and since Fas and Fas ligand are the main apoptotic pathway members, they may represent candidate genes involved in the development of preeclampsia. A polymorphism at the 670 position (A-G) in the Fas gene has been found more frequently in Hungarian women with preeclampsia. STUDY DESIGN The study cohort was a group of 50 women with preeclampsia and 142 healthy control subjects from the general Italian population. They were studied, by RFLP analysis, to validate the role that the 670 G Fas gene polymorphism plays in preeclampsia, and to evaluate the Fas ligand IVS2nt 124 G polymorphism. The Fishers exact test was used to compute the statistical difference between groups. RESULTS The presence of the 670 G Fas gene variant was observed in 42 preeclamptic patients (84%) and 96 members of the general population control group (67.6%) (p=0.029). Regarding the Fas ligand gene, the IVS2nt 124 G variant was present in 14 preeclamptic patients (28%) and in 47 of the general population control subjects (33.1%) (p=0.6). CONCLUSIONS The present study validated the hypothesis that the Fas 670 G variant may have an influencing role in preeclampsia.