Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females.

Droplet-digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)-affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females (n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [SD]) copy/104 cells in SA and 3.02 ( ± 1.86 [SD]) copy/104 cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [SD]) copy/104 cells and 4.09 ( ± 4.26 [SD]) copy/104 cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA- and 2/5 (40%) VI-MCPyV-positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples.

Investigation on silent bacterial infections in specimens from pregnant women affected by spontaneous miscarriage.

Miscarriage is one of the main complications occurring in pregnancy. The association between adverse pregnancy outcomes and silent bacterial infections has been poorly investigated. Ureaplasma parvum and urealiticum, Mycoplasma genitalium and hominis and Chlamydia trachomatis DNA sequences have been investigated by polymerase chain reaction (PCR) methods in chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from females with spontaneous abortion (SA, n = 100) and females who underwent voluntary interruption of pregnancy (VI, n = 100). U. parvum DNA was detected in 14% and 15% of SA and VI, respectively, with a mean of bacterial DNA load of 1.3 × 10-1 copy/cell in SA and 2.8 × 10 -3 copy/cell in VI; U. urealiticum DNA was detected in 3% and 2% of SA and VI specimens, respectively, with a mean DNA load of 3.3 × 10-3 copy/cell in SA and 1.6 × 10-3 copy/cell in VI; M. hominis DNA was detected in 5% of SA specimens with a DNA load of 1.3 × 10-4 copy/cell and in 6% of VI specimens with a DNA load of 1.4 × 10-4 copy/cell; C. trachomatis DNA was detected in 3% of SA specimens with a DNA load of 1.5 × 10-4 copy/cell and in 4% of VI specimens with a mean DNA load of 1.4 × 10-4 copy/cell. In PBMCs from the SA and VI groups, Ureaplasma spp, Mycoplasma spp and C. trachomatis DNAs were detected with a prevalence of 1%-3%. Bacteria were investigated, for the first time, by quantitative real-time PCR (qPCR) in chorionic villi tissues and PBMCs from women affected by SA and VI. These data may help to understand the role and our knowledge of the silent infections in SA.

Vaginal Lactoferrin Modulates PGE2, MMP-9, MMP-2, and TIMP-1 Amniotic Fluid Concentrations.

Inflammation plays an important role in pregnancy, and cytokine and matrix metalloproteases (MMPs) imbalance has been associated with premature rupture of membranes and increased risk of preterm delivery. Previous studies have demonstrated that lactoferrin (LF), an iron-binding protein with anti-inflammatory properties, is able to decrease amniotic fluid (AF) levels of IL-6. Therefore, we aimed to evaluate the effect of vaginal LF administration on amniotic fluid PGE2 level and MMP-TIMP system in women undergoing genetic amniocentesis. One hundred and eleven women were randomly divided into controls (n = 57) or treated with LF 4 hours before amniocentesis (n = 54). Amniotic fluid PGE2, active MMP-9 and MMP-2, and TIMP-1 and TIMP-2 concentrations were determined by commercially available assays and the values were normalized by AF creatinine concentration. PGE2, active MMP-9, and its inhibitor TIMP-1 were lower in LF-treated group than in controls (p < 0.01, p < 0.005, and p < 0.001, resp.). Conversely, active MMP-2 (p < 0.0001) and MMP-2/TIMP-2 molar ratio (p < 0.001) were increased, whilst TIMP-2 was unchanged. Our data suggest that LF administration is able to modulate the inflammatory response following amniocentesis, which may counteract cytokine and prostanoid imbalance that leads to abortion. This trial is registered with Clinical Trial number NCT02695563.

Doppler velocimetry of the ovarian artery as a tool to detect LH surge in stimulated cycles.

Our aim was to assess the velocimetric pattern of the ovarian artery as a possible marker of LH surge in stimulated cycles. A total of 130 women undergoing ovarian stimulation for intrauterine insemination were randomized in two groups. Each woman was stimulated with 75 IU of recombinant FSH starting from the third day of the cycle. Velocimetric indices of the dominant ovarian artery were compared between patients with spontaneous LH surge and those needing HCG administration to trigger dominant follicle rupture. The pulsatility index and the ratio between peak systolic flow and lowest diastolic flow were significantly higher in women that had a spontaneous triggering of ovulation. These parameters had a high and very significant positive correlation with the dosage of luteinizing hormone. Threshold values of 2.60 for PI and 7.68 for S/D had a high sensitivity and specificity to predict LH surge. These velocimetric results demonstrated that an increased resistance in the dominant ovarian artery is correlated to LH surge in stimulated cycles. It may represent a sign of relevant clinical utility in timing of intrauterine insemination and/or natural intercourse.

Vaginal lactoferrin administration before genetic amniocentesis decreases amniotic interleukin-6 levels.

AIM: To verify the eventual efficacy of lactoferrin (LF), an iron-binding glycoprotein, to decrease the amniotic concentration of interleukin-6 (IL-6). METHODS: We prospectively enrolled 60 Caucasian patients at the 16th week of their singleton physiological gestation. A vaginal compound containing 300 mg of LF was administered randomly 4 or 12 h prior to amniocentesis, as to obtain 3 groups: A, 20 untreated patients; B, 20 treated 4 h before amniocentesis; C, 20 treated 12 h before amniocentesis. RESULTS: A normal karyotype was registered in all cases. The comparison of the distribution of IL-6 among the 3 groups showed a highly significant difference (p = 0.001). The difference between mean values of group B and both groups C and A was shown to be highly significant (p = 0.006 and p = 0.03, respectively). In contrast, there was no significant difference between mean values of groups A and C. CONCLUSION: Vaginal LF administration decreases amniotic IL-6 concentration. We therefore suggest that the glycoprotein may exert a protective role against ominous pregnancy complications linked to an increased level of the cytokine, such as abortion secondary to amniocentesis.

Rare case of massive congenital bilateral chylothorax in a hydropic fetus with true mosaicism 47,XXX/46,XX.

Fetal congenital chylothorax is a rare condition that occurs sporadically or can be associated with abnormal karyotype or structural chromosomal anomalies. We report a unique case of fetal congenital bilateral chylothorax associated with mosaicism 47,XXX/46,XX. A female fetus affected by massive bilateral hydrothorax and ascites was diagnosed at 34(+1) weeks of gestation. Previous ultrasonographic exams were completely normal. Immune causes of hydrops were excluded. Elective cesarean section was performed soon after bilateral thoracocentesis. The analysis of drained pleural fluid revealed its lymphatic nature. The fetal karyotyping, performed on chorionic villi at the 11th week, had shown mosaicism 47,XXX/46,XX, later confirmed in the newborn's blood. We hypothesized that chylothorax may be part of the phenotypic spectrum of 47 XXX karyotype and we suggest an ultrasound follow-up of the fetus at closer intervals than the routine timing for this condition, even if it is not usually characterized by severe phenotypic features.

Can Doppler study of the ovarian artery predict the fertility outcome of intrauterine insemination?

BACKGROUND: To test the velocimetric pattern of the ovarian artery as a routine ovarian reserve test. METHODS: We enrolled 317 consecutive patients from January 2011 to June 2012. At the second day of the menstrual cycle, a transvaginal ultrasound was performed to evaluate the antral follicle count and ovarian volume, and Doppler of both ovarian arteries was also performed. Controlled ovarian stimulation was performed and the patients were divided in two groups according to the result of the intrauterine insemination: group A (nonpregnant women) and group B (pregnant women). RESULTS: Ovarian velocimetric pattern was similar between the two groups. Follicle stimulating hormone value had a significant correlation with the ultrasound markers; however, the multiple regression linear analysis showed that the only independent variables were the antral follicle count (t = -2.74, p = 0.008) and the systolic/diastolic ratio (t = 3.95, p = 0.0005). The best parameters in predicting the pregnancy were the mean ovarian volume, total and partial antral follicle count between 7 and 10 mm, and the mean resistance index (area under the curve: 0.744, 0.671, 0.667, 0.573, respectively). CONCLUSIONS: The Doppler study of the ovarian arteries did not add significant information about the ovarian reserve status. Only the mean resistance index had a significant diagnostic accuracy, but its specificity (53%) is too low to consider it a screening test.

Downregulation of A(1) and A(2B) adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi.

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A(1) and A(2B) expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A(1)AR and A(2A)AR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A(2B). In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A(2B) and A(1)ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A(1) and A(2B)ARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.

Corrigendum: Serum IgG antibodies from pregnant women reacting to mimotopes of Simian virus 40 large T antigen, the viral oncoprotein.

[This corrects the article DOI: 10.3389/fimmu.2017.00411.].

The Inflammatory Cytokine Imbalance for Miscarriage, Pregnancy Loss and COVID-19 Pneumonia.

Pregnancy can be defined a vascular event upon endocrine control. In the human hemo-chorial placentation the chorionic villi penetrate the wall of the uterine spiral arteries, to provide increasing amounts of nutrients and oxygen for optimal fetal growth. In any physiological pregnancy the natural maternal response is of a Th1 inflammatory type, aimed at avoiding blood loss through the arteriolar wall openings. The control of the vascular function, during gestation as in any other condition, is achieved through the action of two main types of prostanoids: prostaglandin E2 and thromboxane on the one hand (for vasoconstriction and coagulation), prostacyclin on the other (for vasodilation and blood fluidification). The control of the maternal immune response is upon the responsibility of the fetus itself. Indeed, the chorionic villi are able to counteract the natural maternal response, thus changing the inflammatory Th1 type into the anti-inflammatory Th2. Clinical and experimental research in the past half century address to inflammation as the leading cause of abortion, pregnancy loss, premature delivery and related pulmonary, cerebral, intestinal fetal syndromes. Increased level of Interleukin 6, Interleukin 1-beta, Tumor Necrosis Factor-alfa, Interferon-gamma, are some among the well-known markers of gestational inflammation. On the other side, COVID-19 pneumonia is a result of extensive inflammation induced by viral replication within the cells of the respiratory tract. As it may happen in the uterine arteries in the absence of an effective fetal control, viral pneumonia triggers pulmonary vascular coagulation. The cytokines involved in the process are the same as those in gestational inflammation. As the fetus breathes throughout the placenta, fetal death from placental thrombosis is similar to adult death from pulmonary thrombosis. Preventing and counteracting inflammation is mandatory in both conditions. The most relevant literature dealing with the above-mentioned concepts is reviewed in the present article.

COVID-19 Is a Multifaceted Challenging Pandemic Which Needs Urgent Public Health Interventions.

Until less than two decades ago, all known human coronaviruses (CoV) caused diseases so mild that they did not stimulate further advanced CoV research. In 2002 and following years, the scenario changed dramatically with the advent of the new more pathogenic CoVs, including Severe Acute Respiratory Syndome (SARS-CoV-1), Middle Eastern respiratory syndrome (MERS)-CoV, and the new zoonotic SARS-CoV-2, likely originated from bat species and responsible for the present coronavirus disease (COVID-19), which to date has caused 15,581,007 confirmed cases and 635,173 deaths in 208 countries, including Italy. SARS-CoV-2 transmission is mainly airborne via droplets generated by symptomatic patients, and possibly asymptomatic individuals during incubation of the disease, although for the latter, there are no certain data yet. However, research on asymptomatic viral infection is currently ongoing worldwide to elucidate the real prevalence and mortality of the disease. From a clinical point of view, COVID-19 would be defined as "COVID Planet " because it presents as a multifaceted disease, due to the large number of organs and tissues infected by the virus. Overall, based on the available published data, 80.9% of patients infected by SARS-CoV-2 develop a mild disease/infection, 13.8% severe pneumonia, 4.7% respiratory failure, septic shock, or multi-organ failure, and 3% of these cases are fatal, but mortality parameter is highly variable in different countries. Clinically, SARS-CoV-2 causes severe primary interstitial viral pneumonia and a "cytokine storm syndrome", characterized by a severe and fatal uncontrolled systemic inflammatory response triggered by the activation of interleukin 6 (IL-6) with development of endothelitis and generalized thrombosis that can lead to organ failure and death. Risk factors include advanced age and comorbidities including hypertension, diabetes, and cardiovascular disease. Virus entry occurs via binding the angiotensin-converting enzyme 2 (ACE2) receptor present in almost all tissues and organs through the Spike (S) protein. Currently, SARS-CoV-2 infection is prevented by the use of masks, social distancing, and improved hand hygiene measures. This review summarizes the current knowledge on the main biological and clinical features of the SARS-CoV-2 pandemic, also focusing on the principal measures taken in some Italian regions to face the emergency and on the most important treatments used to manage the COVID-19 pandemic.

Investigation on Spontaneous Abortion and Human Papillomavirus Infection.

Viral infections are considered to be risk factors for spontaneous abortion (SA). Conflicting results have been reported on the association between Human Papillomavirus (HPV) and SA. HPV DNA was investigated in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from women who experienced SA (n = 80, cases) and women who underwent a voluntary interruption of pregnancy (VI; n = 80, controls) by qualitative PCR and quantitative droplet digital PCR (ddPCR). Viral genotyping was performed using real-time PCR in HPV-positive samples. Specific IgG antibodies against HPV16 were investigated in sera from SA (n = 80) and VI (n = 80) females using indirect ELISA assays. None of the DNA samples from SA subjects was HPV-positive (0/80), whilst HPV DNA was detected in 2.5% of VI women (p > 0.05), with a mean viral DNA load of 7.12 copy/cell. VI samples (n = 2) were found to be positive for the HPV45 genotype. The ddPCR assay revealed a higher number of HPV-positive samples. HPV DNA was detected in 3.7% and 5% of SA and VI chorionic tissues, respectively, with mean viral DNA loads of 0.13 copy/cell in SA and 1.79 copy/cell in VI (p >0.05) samples. All DNA samples from the PBMCs of SA and VI females tested HPV-negative by both PCR and ddPCR. The overall prevalence of serum anti-HPV16 IgG antibodies was 37.5% in SA and 30% in VI (p > 0.05) women. For the first time, HPV DNA was detected and quantitatively analyzed using ddPCR in chorionic villi tissues and PBMCs from SA and VI women. Circulating IgG antibodies against HPV16 were detected in sera from SA and VI females. Our results suggest that HPV infection in chorionic villi may be a rare event. Accordingly, it is likely that HPV has no significant role in SA.

Mother-to-child transmission of oncogenic polyomaviruses BKPyV, JCPyV and SV40.

OBJECTIVES: Polyomavirus (PyV) infections have been associated with different diseases. BK (BKPyV), JC (JCPyV) and simian virus 40 (SV40) are the three main PyVs whose primary infection occurs early in life. Their vertical transmission was investigated in this study. METHODS: PyV sequences were analyzed by the digital droplet PCR in blood, serum, placenta, amniotic fluid, vaginal smear from two independent cohorts of pregnant females and umbilical cord blood (UCB) samples. IgG antibodies against the three PyVs were investigated by indirect E.L.I.S.As with viral mimotopes. RESULTS: DNAs from blood, vaginal smear and placenta tested BKPyV-, JCPyV- and SV40-positive with a distinct prevalence, while amniotic fluids were all PyVs-negative. A prevalence of 3%, 7%, and 3% for BKPyV, JCPyV and SV40 DNA sequences, respectively, was obtained in UCBs. Serum IgG antibodies from pregnant females reached an overall prevalence of 62%, 42% and 17% for BKPyV, JCPyV and SV40, respectively. Sera from newborns (UCB) tested IgG-positive with a prevalence of 10% for BKPyV/JCPyV and 3% for SV40. CONCLUSIONS: In this investigation, PyV vertical transmission was revealed by detecting PyV DNA sequences and IgG antibodies in samples from females and their offspring suggesting a potential risk of diseases in newborns.

Influence of obstetric factors on osteogenic potential of umbilical cord-derived mesenchymal stem cells.

Wharton's jelly from the umbilical cord is a noncontroversial source of mesenchymal stem cells (WJMSCs) with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. WJMSCs from different donors have been characterized for their osteogenic potential. Although there is large evidence of WJMSCs plasticity, recently scientific debate has focused on MSCs selection, establishing predictable elements to discriminate the cells with most promising osteoprogenitor cell potential.In the present study a comparative study between the presence of osteoblastic markers and different parameters that pertain to both the newborn and the mother was performed. Umbilical cords were collected after all patients signed the informed consent and local ethical commettee approved the study. Obstetric parameters, including baby's gender and birth weight, mother's age at delivery, gestational stage at parturition and mode of delivery were examined. After characterization and expansion, WJMSCs were analyzed for two osteoblastic markers, alkaline phosphatase (ALP) activity, and the expression level of RUNX-2 transcription factor, and for their ability to deposit mineralized matrix after osteogenic induction.We found that osteoblastic potential was not influenced by baby's gender and mode of delivery. On the contrary, the highest degree of osteoblastic potential has been shown by WJMSCs with RUNX-2 high basal levels, selected from umbilical cords of the heaviest term babies.Even if further evaluation is required, our hypothesis is that our findings may help in selecting the optimal umbilical cord donors and in collecting high potential Wharton's jelly-derived osteoprogenitors efficiently.

Human osteoclasts differentiated from umbilical cord blood precursors are less prone to apoptotic stimuli than osteoclasts from peripheral blood.

Osteoclasts (OCs) are specialized bone-resorbing cells. For "in vitro" analysis, they may be obtained from the precursors present in peripheral blood (PB) or umbilical cord blood (UCB), but there has been no detailed analysis of how the kind of source and cell culture conditions may affect the behavior of these cells. Here we analyzed the behavior of OCs after transfection with specific transcription factor decoy molecules founding that the OCs from PB undergo apoptosis when nuclear factor kappa B (NF-kB) or NFATc1 were removed, or when ERalpha expression was increased. Conversely, OCs from UCB showed a strong resistance to apoptotic stimuli. We found that survival signals including Bcl-2, Bcl-XL, and Survivin are present in the OCs/UCB, but not in OCs/PB. The resistance to apoptosis seems to be not correlated with NF-kB, NFATc1, or ERalpha expression level, or with the activation of ERK and Akt proteins. One of the mechanisms responsible for bone remodeling is apoptosis, and being susceptible of therapeutic manipulation, the OCs are extensively employed to investigate cell response to therapies for the treatment of bone loss associated with several diseases, including periodontitis, osteoporosis, and metastatic osteolysis. Therefore, our evidences are to be taken in consideration when both the effects of biological modifiers are tested and OCs apoptosis molecular mechanisms are investigated.

Control of human trophoblast function.

The trophoblast, i.e. the peripheral part of the human conceptus, exerts a crucial role in implantation and placentation. Both processes properly occur as a consequence of an intimate dialogue between fetal and maternal tissues, fulfilled by membrane ligands and receptors, as well as by hormone and local factor release. During blastocyst implantation, generation of distinct trophoblast cell types begins, namely the villous and the extravillous trophoblast, the former of which is devoted to fetal-maternal exchanges and the latter binds the placental body to the uterine wall. Physiological placentation is characterized by the invasion of the uterine spiral arteries by extravillous trophoblast cells arising from anchoring villi. Due to this invasion, the arterial structure is replaced by amorphous fibrinoid material and endovascular trophoblastic cells. This transformation establishes a low-resistance, high-capacity perfusion system from the radial arteries to the intervillous space, in which the villous tree is embedded. The physiology of pregnancy depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a derangement of such processes can lead to different types of complications of varying degrees of gravity, including possible pregnancy loss and maternal life-threatening diseases. In this review we describe the mechanisms which regulate trophoblast differentiation, proliferation, migration and invasiveness, and the alterations in these mechanisms which lead to pathological conditions. Furthermore, based on the growing evidence that proper inflammatory changes and oxidative balance are needed for successful gestation, we explain the mechanisms by which agents able to influence such processes may be useful in the prevention and treatment of pregnancy disorders.

Influence of vaginal lactoferrin administration on amniotic fluid cytokines and its role against inflammatory complications of pregnancy.

BACKGROUND: An altered amniotic cytokine profile has been reported in inflammatory pregnancy complications with a leading role for IL-6, a marker of the foetal systemic inflammatory response. Up to this date there is no exhaustive information neither on the foetal cytokine balance nor on the best method for its modulation. We aimed to evaluate the influence of vaginal lactoferrin administration on amniotic fluid concentration of 47 cytokines, chemokines and growth factors. METHODS: Sixty women undergoing genetic amniocentesis were enrolled in an open-label clinical trial. 300 mg of vaginal lactoferrin (Florence, Italy) were randomly administered to obtain 3 groups: A, 20 untreated patients; B and C (20 patients in each) respectively treated 4 and 12 h before amniocentesis. Cytokines, chemokines and growth factors concentrations were quantified by a magnetic bead Luminex multiplex immunoassays panel technology. Data analysis was performed with the software Stata (v. 13.1) and GraphPad Prism (v. 5). Group comparisons were performed using Kruskal-Wallis followed by Mann-Whitney U tests, with Bonferroni correction for multiple comparisons. A p < 0.05 was considered significant. RESULTS: Among the 47 tested mediators, 24 (51.06%) were influenced by lactoferrin. 11 (23.4%), showed a highly significant difference (p <0.001); among these IL-9, IL-15, IFN-γ, IP-10, TNF-α, IL-1α and MCP-3 underwent a down-regulation, while IL-17 and FGF-basic, G-CSF, GM-CSF an up-regulation. Difference between group C and both B and A was small for IL-15, IP-10, IL-1α, MCP-3, while it was negligible for IL-9, IFN-γ and TNF-α. IL-17 and the 3 growth factors were strongly enhanced in B and C groups. IL-17, FGF-basic and GM-CSF showed increasing concentrations in both B and C groups, while G-CSF resulted up-regulated only in group C. Significance was intermediate (p < 0.01) for the down regulated IL-2RA, IL-12p40 and IFNα2 (6.38%) while it was small for 10 mediators (21.27%) 7 of which (IL-2, IL-4, eotaxin, PDGF-BB, RANTES, IL-18 and MIF) down-regulated and 3 (MCP-1, IL-3, and SDF-1α) up-regulated. CONCLUSION: Lactoferrin down-regulates 17 pro-inflammatory amniotic mediators while up-regulating 7 anti-inflammatory amniotic mediators, 5 of which definitively belonging to an anti-inflammatory profile. These findings open to clinical investigation on its use against inflammatory complications of pregnancy.

Serum IgG Antibodies from Pregnant Women Reacting to Mimotopes of Simian Virus 40 Large T Antigen, the Viral Oncoprotein.

Simian virus 40 (SV40) large T antigen (LT) coding sequences were revealed in different human samples, whereas SV40 antibodies (Ab) were detected in human sera of cancer patients and healthy individuals, although with a lower prevalence. Previous studies carried out by the neutralization assay gave a SV40 seroprevalence, in the general population, up to 8%, although higher rates, 12%, were detected in kidney transplant children, in a group of HIV-positive patients, and in healthy females. In this study, serum samples from pregnant women, together with those from non-pregnant women, were analyzed to check the prevalence of IgG Ab reacting to SV40 LT antigens. Serum samples were collected from pregnant and non-pregnant women, with the same mean age. Women were in the range of 15-48 years old. Samples were assayed by an indirect ELISA employing specific SV40 LT mimotopes as antigens, whereas functional analysis was performed by neutralization of the viral infectivity in cell cultures. As a control, sera were analyzed for Ab against BK polyomavirus (BKPyV), which is a human polyomavirus homologous to SV40. Statistical analyses employed chi-square with Yates' correction, and Student's t tests. Indirect ELISAs indicated that pregnant women tested SV40 LT-positive with a prevalence of 17% (23/134), whereas non-pregnant women had a prevalence of 20% (36/180) (P > 0.05). Ab against BKPyV were detected with a prevalence of 80% in pregnant women and with a prevalence of 78% in non-pregnant women. These data indicate that SV40 infects at a low prevalence pregnant women. We may speculate that SV40, or a close human polyomavirus still undetected, could be transmitted from mother to fetus.

The role and modulation of the oxidative balance in pregnancy.

Oxidative processes exert a fundamental regulatory function during pregnancy. It depends on the influence of oxygen, nitric oxide, reactive oxygen species and reactive nitrogen species metabolic pathways upon the vascular changes in the maternal organism, as well as on the regulation of uterine and cervical tone throughout gestation and delivery. These functions are strictly linked with the mediators of the inflammatory pathway. At the beginning of pregnancy, when a certain grade of inflammatory change is necessary to the trophoblast invasion of maternal tissue, the activation of the process by nitric oxide and reactive nitrogen species is welcome. Indeed, these products modulate the metalloproteinases, which are responsible for the remodelling of uterine extracellular matrix. At this stage estrogens are involved as well in the regulation of the delicate balance of pro-oxidant and anti-oxidant effects. Furthermore, reactive oxygen and nitrogen species appear to play an important role both in normal and pathologic embryogenesis. During advanced pregnancy, a derangement of the oxidative balance can lead to the improper activation of inflammatory changes, thus triggering premature labour as well as other complications, such as foetal growth restriction and preeclampsia. Although a number of pro- and anti-oxidant agents are available to influence the above-mentioned processes, there is no way to adequately measure the oxidative needs in single cases, in order to modulate the oxidative balance in clinical practice. Pharmacological research should be addressed to the development of new drugs, as well as to selective methods of delivery to the gestational tissues.

Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour.

OBJECTIVES: The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca2+]i) during physiological pregnancy and labour. METHODS: Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement. RESULTS: fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca2+]i. CONCLUSIONS: These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.