Footprints of BK and JC polyomaviruses in specimens from females affected by spontaneous abortion.

STUDY QUESTION: Are JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) infections associated with spontaneous abortion (SA)? SUMMARY ANSWER: There is no association of JCPyV or BKPyV with SA. WHAT IS KNOWN ALREADY: A large number of risk factors have been associated with SA. The role of polyomaviruses, including JCPyV and BKPyV, in SA remains to be clarified. STUDY DESIGN, SIZE, DURATION: This is a case-control study including women affected by spontaneous abortion (SA, n = 100, the cases) and women who underwent voluntary interruption of pregnancy (VI, n = 100, the controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: Viral DNAs were investigated by qualitative PCR and quantitative droplet-digital PCR (ddPCR) in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from SA (n = 100) and VI (n = 100). Indirect ELISAs with mimotopes/synthetic peptides corresponding to JCPyV and BKPyV viral capsid protein 1 epitopes were then employed to investigate specific IgG antibodies against JCPyV and BKPyV in human sera from SA (n = 80) and VI (n = 80) cohorts. MAIN RESULTS AND THE ROLE OF CHANCE: JCPyV DNA was detected in 51% and 61% of SA and VI samples, respectively, with a mean viral DNA load of 7.92 copy/104 cells in SA and 5.91 copy/104 cells in VI (P > 0.05); BKPyV DNA was detected in 11% and 12% of SA and VI specimens, respectively, with a mean viral DNA load of 2.7 copy/104 cells in SA and 3.08 copy/104 cells in VI (P > 0.05). JCPyV was more prevalent than BKPyV in both SA and VI specimens (P < 0.0001). In PBMCs from the SA and VI cohorts, JCPyV DNA was detected with a prevalence of 8% and 12%, respectively, with a mean viral DNA load of 2.29 copy/104 cells in SA and 1.88 copy/104 cells in VI (P > 0.05). The overall prevalence of serum IgG antibodies against JCPyV detected by indirect ELISAs was 52.5% and 48.7% in SA and VI groups, respectively, whereas BKPyV-positive sera were found in 80% SA and 78.7% VI samples. LIMITATIONS, REASONS FOR CAUTION: This study did not investigate the presence of viral mRNA and/or proteins, which are indicative of an active viral infection, and these might be taken into consideration in future studies. WIDER IMPLICATIONS OF THE FINDINGS: JCPyV and BKPyV DNA sequences were detected and quantitatively analyzed for the first time by PCR/ddPCR in chorionic villi tissues and PBMCs from SA and VI specimens. Moreover specific immunological approaches detected serum IgG against JCPyV/BKPyV. Statistical analyses, however, do not indicate an association between these polyomaviruses and SA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University of Ferrara, FAR research grants and the University Hospital of Ferrara/University of Ferrara joint grant. No potential conflicts of interest were disclosed.

Betamethasone, progesterone and RU-486 (mifepristone) exert similar effects on connexin expression in trophoblast-derived HTR-8/SVneo cells.

Connexins (Cx) are membrane proteins able to influence cell trophoblast responses, such as proliferation, differentiation, migration and invasiveness. Likewise, glucocorticoids are also known to modulate many factors involved in implantation, including trophoblast gap-junction intercellular communication, although their influence on pregnancy is controversial. In order to investigate the effects of betamethasone, a synthetic glucocorticoid, on Cx and glucocorticoid receptor (GR) expression and localisation, as well as on cell proliferation, the extravillous trophoblast-derived HTR-8/SVneo cell line was used as a model. The results, confirmed by means of immunofluorescence, demonstrate that betamethasone selectively modifies GR and Cx expression, enhancing the GRα isoform without affecting GRß, and inhibiting Cx40 expression whilst increasing that of Cx43 and Cx45. Furthermore, betamethasone was shown to exert an inhibitory action on cell proliferation. In this model the abortion drug RU-486 (mifepristone), reported to be a GR antagonist, did not counteract this effect of betamethasone. On the contrary, it induced responses similar to those of the hormone. Knowing that RU-486 is also a potent progesterone-receptor antagonist, the effect of progesterone alone and in combination with the drug on Cx expression and cell proliferation was then tested. Progesterone showed the same effect as betamethasone on Cx expression, but it did not affect proliferation. Based on these results, neither the abortion effects of RU-486 nor the protective action of betamethasone and progesterone are exerted by modulation of Cx. RU-486 did not antagonise the progesterone effect, suggesting that its abortive action does not involve alteration of trophoblast Cx expression.

cAMP efflux from human trophoblast cell lines: a role for multidrug resistance protein (MRP)1 transporter.

Cyclic adenosine 3'-5'-monophosphate (cAMP) is a second messenger, which exerts an important role in the control of human first-trimester trophoblast functions. In the present study we demonstrate the existence of a mechanism that is able to extrude cAMP from trophoblast-derived cell lines, and show evidence indicating the involvement of multidrug resistance protein (MRP) 1, a transporter belonging to the ATP-binding cassette family, in cAMP egress. MRP1 is expressed in trophoblast cell lines and cAMP efflux is highly reduced by the MRP1 inhibitor, MK-571. In addition, interleukin-1beta and estrone are able to enhance MRP1 gene expression and influence extracellular cAMP concentration. The occurrence of a MRP1-dependent cAMP efflux is also shown in human first-trimester placenta explants. Extracellular cAMP could represent a source for adenosine formation, which in turn could regulate cAMP-dependent responses in placental tissue. Evidence is provided that adenosine receptor subtypes are present and functional in human trophoblast-derived cells. A role for cAMP egress mechanism in the fine modulation of the nucleotide homeostasis is therefore suggested.

Somatostatin as a regulator of first-trimester human trophoblast functions.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

Prostaglandin E2 inhibits proliferation and migration of HTR-8/SVneo cells, a human trophoblast-derived cell line.

Normal placentation requires a highly coordinated control of proliferation, migration and invasiveness of extravillous trophoblast cells. Since prostaglandin E2 is a major prostanoid synthesized by intrauterine tissues and highly involved in pregnancy homeostasis, we examined the possibility that it modulates extravillous trophoblast cell functions. Here, we report the presence of mRNAs for prostaglandin E2 EP2 and EP4 receptor isoforms and of proteins in both first-trimester human chorionic villi and in the human trophoblast-derived HTR-8/SVneo cells. Moreover we found that: (i) this cell line releases prostaglandin E2 and the output is enhanced by interleukin-1beta; (ii) the prostanoid consistently inhibits serum- or epidermal growth factor-induced cell proliferation and also migration. An involvement of cAMP in the prostaglandin E2 antiproliferative action is suggested by the observation that the prostanoid greatly enhances cAMP level in HTR-8/SVneo cells and that forskolin inhibits cell proliferation; moreover the administration of prostaglandin E2 plus forskolin, a condition which evokes a synergistic enhancement of cAMP, induces a major impairment of cell growth. Provided that our data are applicable to the trophoblast tissue in vivo, we suggest that prostaglandin E2 exerts an important control on extravillous trophoblast cell functions, preventing an excessive proliferation and migration.

Effect of nitric oxide on arachidonic acid release from human amnion-like WISH cells.

In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [(3)H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; (3)L -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3',5'-cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E(2) (PGE(2)) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [(3)H]arachidonic acid release. Both soluble guanylyl cyclase and PLA(2) inhibitors counteracted SNP response. Exogenous NO increased PGE(2) release, although to a much lesser degree compared with arachidonic acid release. Our results indicate that NO stimulates AA release in WISH cells by activating PLA(2) through a cyclic GMP-dependent mechanism.

WISH cells as a model for the "in vitro" study of amnion pathophysiology.

In the course of pregnancy amnion cells produce a number of factors which include cytokines and prostaglandins (PGs) produced in response to autocrine, paracrine and endocrine signals. Recent studies performed by several researchers contributed to elucidate the mechanism through which amnion tissue is involved in the triggering of physiological labor. However, there are other possible functions to be ascribed to amniotic cells, depending on the high number of factors that they produce as well as on the receptors that enable them to act in turn as target. For instance, it has been demonstrated that amnion cells are able to produce lecithin upon the regulation of several factors, such as glucocorticoids and epidermal growth factor, a finding that suggests a protective role of the tissue on fetal pulmonary function. As regards to triggering the uterine contractions, it is accepted that prostaglandin release by amnion cells represents a key event. It is under the control of hormones, growth factors, cytokines and probably PGs themselves. A striking analogy has been found between the mechanism of inflammation and the onset of myometrial activity in labor. In this context, it has been shown that for-Met-Leu-Phe (fMLP), the prototype of a series of formylated peptides traditionally considered chemotactic agents, is also involved in the regulation of amniotic PG release. The similitude between labor and inflammatory response is enforced by the antiprostaglandin action of some classes of antibiotics observed in amnion tissue, that enable them as effective tools against preterm labor, both in the absence and in the presence of infection. As for the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers, mainly represented by phosphatidyl-inositol-4-5 bisphosphate and cyclic AMP. The mechanism whereby second messengers induce PG release is not clear, and a crosstalk between the two transduction pathways could be hypothesized. This interaction has extensively been analysed in "WISH" cells, a human amnion-derived cell line, which represent a model for the in vitro study of amnion functions. In the present review, we intend to report the results of the studies regarding the mechanisms through which the control of the above mentioned functions is executed.

Plasminogen activator system in serum and amniotic fluid of euploid and aneuploid pregnancies.

OBJECTIVE: To compare euploid and aneuploid pregnancies with respect to maternal serum and amniotic fluid (AF) levels of the components of the plasminogen system. METHODS: The study population consisted of 123 single pregnancies at the 17th gestational week, 16 with minor chromosomal abnormalities, 15 aneuploid, and 92 euploid. RESULTS: Both groups with chromosomal abnormalities had significantly higher serum levels of urokinase plasminogen activator and its complexed form with its type-1 inhibitor compared with euploid pregnancies. In AF, tissue plasminogen activator was significantly lower in the aneuploid than the euploid group, whereas type-1 inhibitor of plasminogen activator was significantly higher in the cases with minor chromosomal abnormalities compared with euploid. At cutoff levels set at 100% sensitivity, the complexed form of urokinase plasminogen activator with its type-1 inhibitor had the strongest specificity (66.3%); after logarithmic transformation, its serum level was 7.53 times higher in aneuploidies than euploidies. CONCLUSION: Aneuploid pregnancies appear to be accompanied by abnormalities of the plasminogen activation system, which could lead to impaired placental perfusion and thus to abortion, fetal death, and fetal growth restriction.

Use of the copper intrauterine device in the management of secondary amenorrhea.

OBJECTIVE: To determine whether the insertion of a copper intrauterine device can restore regular menses in patients with functional secondary amenorrhea. DESIGN: Prospective, observational study. SETTING: Clinical practices. PATIENT(S): Forty-eight volunteers with functional secondary amenorrhea. INTERVENTION(S): Insertion of a copper intrauterine device. MAIN OUTCOME MEASURE(S): Restoration of menses. RESULT(S): In 40 patients, regular menses were restored within a few weeks after insertion of the device. Normal menses were maintained as long as the copper intrauterine device remained in place. After removal of the device, normal menses persisted for 1 year. CONCLUSION(S): Insertion of a copper intrauterine device restores regular menses in women with functional secondary amenorrhea. The mechanism of action of the device probably is related to the release of prostaglandins from the endometrium.

Effect of different classes of antibiotics on amniotic prostaglandin E release.

Our purpose was to investigate the effects of different classes of antibiotics, namely beta-lactamines, aminoglicosides, tetracyclines, macrolides, on amniotic prostaglandin E release to clarify their role in the treatment of premature labor. The effects of these antibiotics were tested also in combination with ampicillin, whose antiprostaglandinergic action had been demonstrated previously. Ceftriaxone and gentamicin significantly and reversibly inhibit both basal and arachidonic acid- or oxytocin-stimulated prostaglandin E release from amnion, although to a different extent. On the contrary, tetracycline and erythromycin do not influence prostaglandin E output. The inhibitory effect of ampicillin is potentiated, in an additive manner, by ceftriaxone, reduced by gentamycin, and eliminated by tetracycline and erythromycin. The finding that diverse classes of antibiotics and their combinations affect amniotic prostaglandin E release should be taken into account in the management of premature labor.

Analogs of cord red blood cell membrane components displayed on placenta, amnion and amniotic cells in culture.

Placenta and amnion were analyzed to ascertain the presence of antigens in common with red blood cells (RBC) from cord or fetuses. The expression of distinct antigens displayed on a subpopulation of cord RBC and detected by anticord RBC membrane antibodies was particularly investigated, concomitantly with the presence of transferrin receptors (TR) by employing immunohistochemistry. The placenta showed both cord antigen and TR; on the contrary, amnion--which was labelled by anti-cord RBC membrane antibodies--was not stained by the anti-TR antibody. The results of inhibition and double labelling assays further excluded TR as the relevant antigen in the labelling of both placenta and amnion. The staining of fetal membranes disappeared after absorption of antibodies with cord RBC membranes. These results suggest that the antigens externally expressed on a subpopulation of cord RBC are shared by amnion and placenta.

alpha-L-fucosidase activity in endometrial, cervical and ovarian cancer.

alpha-L-fucosidase activity assayed on endometrial, cervical and ovarian tissue in benign and malignant conditions has been found to be increased in cancer tissue. Knowing that alpha-L-fucose plays a fundamental role in the process of inhibition of macrophages migration, increased activity of fucosidase in tumors can be interpreted as a possible mechanism by which cancer cells directly subvert the process of macrophages activation thus facilitating xenoplastic growth.

"In vitro" effect of progesterone on alpha-L-fucosidase activity of human endometrium.

Alpha-L-fucosidase activity was found to be increased in organ cultured normal endometrium and in endometrial adenocarcinoma compared with non cultured tissue. The enzyme activity was lowered with addition of progesterone to the medium. Non cultured adenocarcinoma showed a higher activity compared with non cultured normal endometrium.

Sonographic picture of submembranous haematoma.

Two cases of vaginal bleeding during pregnancy where elevation of the membranes was found by means of echography are reported. In one case there was an accessory lobe with a marginal haematoma. No evidence of placenta praevia was found. Elevation of the membranes can represent an echographic sign of submembranous haematoma.

Prenatal diagnosis of cystic adenomatoid malformation of the lung.

Two cases of congenital cystic adenomatoid malformation of the lung (CCAML) are described. In the light of recent literature the prenatal diagnosis and management are discussed.

Contribution to the assessment of steroid therapy in the prevention of respiratory distress syndrome in the neonate.

The respiratory distress syndrome (RDS) is a physiological manifestation of neonatal pulmonary immaturity and it is still the major cause of neonatal morbidity and mortality. In order to promote early fetal lung maturity when a preterm delivery is anticipated, a number of pharmacological agents have been investigated. Corticosteroids, in particular, have been extensively used and the results of several trials are reported in literature. A cohort of 246 consecutive singleton preterm infants, liveborn at the Obstetric Clinic of Ferrara University during a 5-year period, was studied to assess whether antenatal steroid therapy reduces the incidence of RDS. Respiratory distress developed in 18.6% of 102 babies who received treatment and in 15.3% of 144 controls, without difference at the statistical analysis. According to previous studies, a lower incidence of RDS was only observed in the treated females compared to non-treated controls (35% vs 46%) at the gestational age of 28-33 weeks. Since the efficacy of steroids seems to be restricted to a very small and specific group of babies, who, moreover are relatively mature by modern intensive care standards, the Authors suggest that the prevention of RDS and its related complications should rely much more on appropriate surveillance and management of the mother and infant than on specific pharmacological interventions.

Amniotic fluid beta 2-microglobulin (beta 2-m) as an index of fetal maturity.

Amniotic fluid beta 2 microglobulin (beta 2-m) levels were measured by radioimmunoassay in 78 pregnant women between the 14th and the 42nd week of gestation. 62 were healthy subjects, while eight were affected by EPH gestosis, seven by diabetes (cl. B-F) associated with Rh immunization in one case, one by hydramnios. There was no significant correlation either between beta 2-m and creatinine (n = 18), or between beta 2-m and lecithin sphingomyelin ratio (L/S) (n = 16), although low concentrations of beta 2-m were usually observed after the 35th week of gestation. It is noteworthy that only in one case out of seven with amniotic levels less than 5 microgram/ml L/S ratio was less than 2.

Adenylate cyclase and cyclic AMP-phosphodiesterase activities of fetal membranes: effect of insulin.

Basal activity and response to insulin of adenylate cyclase and cyclic AMP-phosphodiesterase were measured in the fetal membranes before and after labor. Basal activity of adenylate cyclase showed only slight variations after delivery when compared to that observed before labor. Enzyme activity was significantly although weakly decreased by insulin both in the amnion and in chorion before labor, while after delivery a weaker non-significant inhibition was observed. Basal activity of cyclic AMP-phosphodiesterase was lower after delivery with respect to that observed before labor. Insulin inhibited enzyme activity in all conditions. Cyclic AMP levels in intact tissue were not substantially modified by insulin.

Hemolytic disease of the newborn secondary to anti-Fya and anti-S.

One case of rare maternal-fetal immunization in a patient affected by Cooley's anemia, is reported. The opportunity for a complete characterization of the blood group and for a search for maternal antibodies in patients with a history of multiple blood transfusions is stressed.

Low dose of betamethasone throughout the whole course of pregnancy and fetal growth: a clinical study.

AIM: To assess the eventual influence of low dose betamethasone throughout pregnancy on fetal growth. PATIENTS AND METHODS: 320 patients - admitted to the Section of Obstetrics and Gynecology of Ferrara University from January 2005 to December 2010 - were subdivided in two groups: 160 patients affected by recurrent spontaneous abortion (Group A), treated by low dose of betamethasone (0.5 mg/daily) throughout pregnancy for preventive purposes, 160 patients with physiological pregnancy as control group (Group B). Primary measured outcomes were neonatal biometric parameters such as birth weight, head circumference and neonatal length. Unpaired t-test was used to compare the neonatal biometric parameters. RESULTS: Birth weight, length and circumference head resulted significantly lower in groups treated by GCs. However, excluding bias as pregnancy complicated by diseases, which could affect fetal growth, biometric neonatal parameters were not different between two groups. Furthermore, analyzing the distribution of the value of birth weight we observed that in the group A there were 44 newborns with a weight even higher than fiftieth percentile. CONCLUSIONS: Betamethasone seems not to influence fetal growth. Our analysis demonstrates that fetal growth is influenced by several factors, therefore, homogeneous study population is essential to have convincing results.