Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling.

OBJECTIVE: To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection. METHODS: Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow. RESULTS: An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell. CONCLUSION: Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.

Mitochondrial DNA content: a new potential biomarker for Sudden Infant Death Syndrome.

BACKGROUND: Sudden Infant Death Syndrome (SIDS) occurs in apparently healthy infants and is unpredictable and unexplained despite thorough investigations and enormous research efforts. The hypothesis tested in this case-control study concerns mitochondrial involvement in SIDS occurrence. METHODS: Mitochondrial DNA content (MtDNAcn) was measured in 24 SIDS cerebral cortex samples and 18 controls using real-time PCR. RESULTS: The median (interquartile range) mtDNAcn in SIDS and controls was 2578 (2224-3838) and 1452 (724-2517) copies per nuclear DNA, respectively (P = 0.0001). MtDNAcn values were higher in SIDS victims born to non-smoking parents (n = 7) 4984 (2832-6908) compared to the controls (n = 5) 2020 (478-2386) (P = 0.006). Increased levels of mtDNAcn have been observed in the SIDS cases with mild defects in nuclei not essential for life compared to those found in SIDS cases with severe alterations of respiratory function (P = 0.034) 3571 (2568-5053) (n = 14) 2356 (1909-3132) (n = 8), respectively. CONCLUSIONS: Our study revealed for the first time higher mtDNAcn in the cerebral cortex of the SIDS cases than the controls, indicating metabolic alterations. MtDNAcn plays an important role in compensatory mechanisms against environmental factors affecting human health. Despite the small sample size, mtDNA may prove to be a potential forensic biomarker for autopsied SIDS victims for gaining new insights into the etiology of SIDS. IMPACT: Mitochondrial DNA content evaluated in cerebral cortex samples is higher in SIDS victims than controls. These results represent a novel line of investigation for the etiology of SIDS and could have a significant role in the compensatory mechanism due to environmental factors affecting human health. These findings suggest that the mitochondria are involved in SIDS: mtDNA content may represent a biomarker of this syndrome.

Mitochondrial DNA copy number in peripheral blood: a potential non-invasive biomarker for female subfertility.

PURPOSE: Low mitochondrial DNA (mtDNA) content in oocytes and in cumulus cells is an indicator of poor oocyte quality. Moreover, initial evidence showed a correlation between mtDNA content in cumulus cells and mtDNA copy number in peripheral blood cells. On these bases, we deemed of interest investigating the correlation between mtDNA copy number in peripheral blood and natural fecundity. METHODS: This is a nested case-control study drawn from a prospective cohort of pregnant women referred for routine first trimester screening for aneuploidies (from 11 + 0 to 12 + 6 weeks of gestation) between January 2012 and March 2013 at the "Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico" of Milan, Italy. Cases were subfertile women who attempted to become pregnant for 12-24 months. Controls were the two subsequently age-matched women who became pregnant in less than 1 year. MtDNA was quantified using real-time PCR and normalized to nuclear DNA. RESULTS: One hundred and four subfertile women and 208 controls were selected. The median (IQR) mtDNA copy number was 95 (73-124) and 145 (106-198), respectively (p < 0.001). The area under the ROC curve was 0.73 (95% CI 0.67-0.79) (p < 0.001). The Youden index was 105 mtDNA copy number. The crude OR for subfertility in women with mtDNA copy number below this threshold was 5.72 (95% CI 3.43-9.55). The accuracy of mtDNA copy number assessment in peripheral blood progressively decreased with increasing female age. CONCLUSIONS: Low mtDNA copy number in peripheral blood is associated with an increased risk of subfertility and may represent a biomarker of natural fecundity.

Serum Levels of 25-Hydroxyvitamin D and Time to Natural Pregnancy.

AIM: The aim of this study was to evaluate whether natural fertility is related to serum 25-hydroxyvitamin D (25-OH-vitamin D) levels. METHODS: A nested case-control study was designed from a prospective cohort of pregnant women undergoing first trimester screening for aneuploidies. Cases included women seeking pregnancy for 12-24 months. Controls were the subsequent age-matched women conceiving in less than 1 year. We excluded women aged ≥40 or <18 years, those assuming supplementary products that included vitamin D before or during pregnancy, those with irregular menstrual cycles or known causes of subfertility, those conceiving through assisted reproductive techniques or requiring ovarian stimulation and those who were overweight or obese. A quantitative detection of serum 25-OH-vitamin D and patients' interview were performed. RESULTS: Seventy-three cases and 73 matched controls were selected. The mean ± SD serum 25-OH-vitamin D was 21.2 ± 6.8 and 19.7 ± 7.3 ng/ml, respectively (p = 0.16). The number (%) of women with serum levels <20 ng/ml (vitamin D insufficiency) was 34 (47%) and 37 (51%), respectively (p = 0.73). The adjusted OR of longer time to pregnancy in women with vitamin D insufficiency was 0.84 (95% CI 0.42-1.66). CONCLUSIONS: Our study does not support a crucial role of 25-OH-vitamin D in natural fertility.

Fimbrial cells exposure to catalytic iron mimics carcinogenic changes.

OBJECTIVE: Recent evidence strongly suggests that the fallopian tube is a site of origin of ovarian cancer. Although histological data show iron deposition in the fallopian tubes, its role remains unclear. To establish whether catalytic iron has a possible role in ovarian carcinogenesis, we isolated human fimbrial secretory epithelial cells (FSECs). METHODS: Fimbrial secretory epithelial cells, isolated from women undergoing isteroannessiectomy, were treated with different doses of catalytic iron (0.05-100 mM) to study cell viability; NO production; p53, Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc protein expressions through Western blot analysis; and immunocytochemistry or immunofluorescence. RESULTS: In FSECs treated with catalytic iron for up to 6 days, we observed an increase in cell viability, NO production, and p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc activations (P < 0.05) in a dose-dependent and time-dependent manner. These same results were also observed in FSECs maintained for respectively 2 and 4 weeks in the absence of catalytic iron after 6 days of stimulation. CONCLUSIONS: Our model aimed at studying the main nongenetic risk factor for ovarian cancer, providing an alternative interpretation for the role of menstruation in increasing risk of this pathology. This in vitro model mimics several features of the precursor lesions and opens new scenarios for further investigations regarding the correlation between damages produced by repeated retrograde menstruation carcinogenic stimuli.

Serum anti-Müllerian hormone in subfertile women.

INTRODUCTION: The correlation between ovarian reserve and infertility remains unclear. Albeit poorly predictive of pregnancy success in in vitro fertilization cycles, serum anti-Müllerian hormone (AMH) has been acknowledged as a surrogate measure of ovarian reserve and is commonly evaluated in women seeking pregnancy. Disentangling whether low serum AMH affects natural fecundity is clinically important, as this information helps physicians in providing appropriate counseling to women and may impact on management strategies. MATERIAL AND METHODS: This was a nested case-control study from a prospective cohort of pregnant women undergoing first trimester screening for aneuploidies. Cases were subfertile women having tried to become pregnant for 12-24 months. Controls were subsequent age-matched fertile women. Inclusion criteria for both cases and controls were: (i) age > 18 years, (ii) natural conception, (iii) regular menstrual cycles (24-35 days). We used quantitative detection of serum AMH and interviews with the women. The main outcome measure was the proportion of women with serum AMH < 1.1 ng/mL. RESULTS: Seventy-six subfertile women and 76 matched fertile controls were selected. In the two study groups, there were 11 (15%) and 15 (20%) women with serum AMH < 1.1 ng/mL, respectively (p = 0.52). The crude odds ratio for subfertility in women with low serum AMH was 0.69 [95% confidence interval (CI) 0.29-1.62]. The adjusted odds ratio was 0.85 (95% CI 0.35-2.10). The median (interquartile range) serum concentration of AMH in subfertile and control women was 2.6 (range 1.6-4.0) and 2.8 (range 1.4-4.3) ng/mL, respectively (p = 0.91). CONCLUSIONS: Low serum AMH is not associated with female subfertility.

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation.

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

A role for mitochondria in gestational diabetes mellitus?

Mitochondrial activity is critical for maintenance of correct glucose homeostasis and alteration in mitochondrial content or function may progressively lead to the development of insulin resistance. Evidence on the possible role of mitochondria in the pathogenesis of gestational diabetes mellitus (GDM) is conversely scanty and inconsistent. The aim was to evaluated mitochondrial DNA (mtDNA) content in peripheral blood of pregnant women with GDM. We selected 25 pregnant women affected by GDM and 50 controls with physiological pregnancies. A blood sample was collected at 32-36 weeks' gestation, stored and thawed simultaneously. The mtDNA content was determined utilizing a quantitative real-time polymerase chain reaction by the Taqman method, using a genomic control and a target gene. Results are expressed as copy number per nuclear DNA. The median (interquartile range) mtDNA content in GDM and controls was 122 (107-198) and 170 (129-196), respectively (p = 0.039). The mtDNA content was also correlated to GDM treatment, self-blood glucose monitoring and newborns' weight, but these analyses failed to document any statistically significant association. Attenuated mitochondrial function may play a role in the development of GDM. Further experiments are required to definitely clarify whether this defect represents a primary event in the pathogenesis of the disease.

From pandemic to syndemic: microbiota, pregnancy, and environment at a crossroad.

Aim: SARS-CoV2 is the latest pandemic that have plagued the socio-health system as an epiphenomenon resulting from planetary resources abuse, crucial for biodiversity. The Anthropocene best defines the present epoch in which human activity irreversibly manipulates intricate and delicate geological and biological balances established over eons. The devastating ecological and socio-economic implications of COVID-19, underline the importance of updating the present pandemic framework to a syndemic. This paper stems from the need to suggest to scientists, doctors, and patients a mission that integrates responsibility from individual to collective health, from present to trans-generational, from human to the entire biotic network. Today's choices are crucial for the perspective on all levels: political, economic, and health as well as cultural.Methods: Research on PubMed and other specific web-sites journal was performed on the topic "Microbiota", "Covid-19", "Pandemic", "Zoonosis", "SARS-CoV-2", "Environmental Pollutants", "Epigenetics", "Fetal Programming", "Human Extinction". Data collected were analysed for an integrative model of interconnection between environment, pregnancy, SARS-CoV-2 infection, and microbiota. Moreover, systematic literature review allowed to summarise in a table information about the worst pandemics that afflicted the human species recently.Results: This paper offers a broad view of the current pandemic starting with pregnancy, the moment when a new life begins and the health trajectories of the unborn child are defined, which will inevitably have repercussions on his well-being. The fundamental role of the biodiversity-rich microbiota in avoiding the development of severe infectious diseases, is therefore highlighted. It is imperative to adjust the current reductionist paradigm based on mostly immediate symptom management towards a broader understanding of the spatial interconnection of ecological niches with human health and the impacts of today's choices on the future. Health and healthcare are elitist rather than egalitarian, therefore focusing on environmental health forces us to make a concerted and systemic effort that challenges political and economic barriers, which are biologically senseless. A healthy microbiota is essential to well-being, both by preventing chronic degenerative conditions, the infectiousness and pathogenicity of bacterial and viral diseases. SARS-CoV-2 should not be an exception. The human microbiota, forged by the first 1,000 days of life, is fundamental in shaping the health-disease trajectories, and by the everlasting exposome that is dramatically affected by the ecological disaster. Individual health is one world health whereas single and global well-being are interdependent in a space-time perspective.Conclusions: Is it not a convenient reductionism not to consider the COVID-19 emergency as a bio-social epiphenomenon of a far more devastating and multi-faceted crisis whose common denominator is the global biotic network loss of which humans are still part?

Mitochondrial DNA Copy Number Drives the Penetrance of Acute Intermittent Porphyria.

No published study has investigated the mitochondrial count in patients suffering from acute intermittent porphyria (AIP). In order to determine whether mitochondrial content can influence the pathogenesis of porphyria, we measured the mitochondrial DNA (mtDNA) copy number in the peripheral blood cells of 34 patients and 37 healthy individuals. We found that all AIP patients had a low number of mitochondria, likely as a result of a protective mechanism against an inherited heme synthesis deficiency. Furthermore, we identified a close correlation between disease penetrance and decreases in the mitochondrial content and serum levels of PERM1, a marker of mitochondrial biogenesis. In a healthy individual, mitochondrial count is usually modulated to fit its ability to respond to various environmental stressors and bioenergetic demands. In AIP patients, coincidentally, the phenotype only manifests in response to endogenous and exogenous triggers factors. Therefore, these new findings suggest that a deficiency in mitochondrial proliferation could affect the individual responsiveness to stimuli, providing a new explanation for the variability in the clinical manifestations of porphyria. However, the metabolic and/or genetic factors responsible for this impairment remain to be identified. In conclusion, both mtDNA copy number per cell and mitochondrial biogenesis seem to play a role in either inhibiting or promoting disease expression. They could serve as two novel biomarkers for porphyria.

Human umbilical cord blood cells suffer major modification by fixatives and anticoagulants.

Introduction: Developing techniques for the tagless isolation of homogeneous cell populations in physiological-like conditions is of great interest in medical research. A particular case is Gravitational Field-Flow Fractionation (GrFFF), which can be run avoiding cell fixation, and that was already used to separate viable cells. Cell dimensions have a key role in this process. However, their dimensions under physiological-like conditions are not easily known since the most diffused measurement techniques are performed on fixed cells, and the fixation used to preserve tissues can alter the cell size. This work aims to obtain and compare cell size data under physiological-like conditions and in the presence of a fixative. Methods: We developed a new protocol that allows the analysis of blood cells in different conditions. Then, we applied it to obtain a dataset of human cord blood cell dimensions from 32 subjects, comparing two tubes with anticoagulants (EDTA and Citrate) and two tubes with different preservatives (CellRescue and CellSave). We analyzed a total of 2071 cells by using confocal microscopy via bio-imaging to assess dimensions (cellular and nuclear) and morphology. Results: Cell diameter measured does not differ when using the different anticoagulants, except for the increase reported for monocyte in the presence of citrate. Instead, cell dimensions differ when comparing anticoagulants and cell preservative tubes, with a few exceptions. Cells characterized by high cytoplasm content show a reduction in their size, while morphology appears always preserved. In a subgroup of cells, 3D reconstruction was performed. Cell and nucleus volumes were estimated using different methods (specific 3D tool or reconstruction from 2D projection). Discussion: We found that some cell types benefit from a complete 3D analysis because they contain non-spherical structures (mainly for cells characterized by poly-lobated nucleus). Overall, we showed the effect of the preservatives mixture on cell dimensions. Such an effect must be considered when dealing with problems highly dependent on cell size, such as GrFFF. Additionally, such information is crucial in computational models increasingly being employed to simulate biological events.

Maternal blood mitochondrial DNA content during normal and intrauterine growth restricted (IUGR) pregnancy.

OBJECTIVE: We investigated mitochondrial DNA (mtDNA) content in the maternal circulation of normal pregnancies of different gestational ages and in pregnancies complicated by intrauterine growth restriction (IUGR). STUDY DESIGN: We examined 70 maternal blood samples: 13 nonpregnant women; 45 normal pregnancies, divided into the 3 trimesters; and 12 pregnancies complicated by IUGR. MtDNA content was determined by real-time quantitative polymerase chain reaction, using a genomic control and a target gene. RESULTS: A highly significant progressive reduction in circulating mtDNA was observed in pregnant women of first, second, and third trimesters and compared to nonpregnant women (mean value: 237, 188, 144, and 283, respectively; P < .001). Moreover, mtDNA was significantly increased in women carrying IUGR fetuses compared to women with normal pregnancies (430 vs 144; P < .001). CONCLUSION: MtDNA could provide new insight into the mechanisms that occur during physiological gestation. Furthermore, mtDNA content may help recognize the IUGR disease in pregnancy.

Procedure for rapid oocyte selection based on quantitative analysis of cumulus cell gene expression.

PURPOSE: To develop a procedure for the analysis of gene expression in cumulus cells during the interval between ovum pick up and insemination to select the best oocytes for fertilization. METHODS: Five RNA extraction methods, three reverse transcription procedures followed by Real-time quantitative PCR and one single-step mRNA quantification kit were tested to measure the expression of five genes in cumulus cells. RESULTS: Two RNA extraction kits gave the best combination of efficiency and purity. One reverse transcription procedure gave the best speed and efficiency. The single-step kit required more biological material than would be available from single cumulus oocyte complexes (COCs). CONCLUSIONS: Our test identified a combination of RNA extraction and reverse transcription procedures that enables the level measurement of 5 selected cumulus cell transcripts within 4 h. Using this combination it was possible to obtain a reliable quantification of gene expression in 44 out of 46 individual COCs collected from seven patients.

Mitochondrial DNA Copy Number in Peripheral Blood in the First Trimester of Pregnancy and Different Preeclampsia Clinical Phenotypes Development: A Pilot Study.

Inflammation and oxidative stress are intrinsically linked to early poor placentation, typical of pregnancies complicated by preeclampsia associated with intrauterine growth restriction (PE-IUGR). Low mitochondrial DNA copy number (mtDNAcn) in peripheral blood constitutes a good peripheral surrogate marker of inflammation and oxidative stress. On these basis, we explored a possible correlation between mtDNAcn in peripheral blood in the first trimester of pregnancy and the PE-IUGR onset. To shed light on this issue, we setup a nested case-control study from a prospective cohort of pregnant women undergoing first-trimester aneuploidies screening. Two groups of patients affected by PE classified according to the clinical phenotype were identified: (1) patients who developed PE-IUGR and (2) patients who developed PE associated with appropriate for gestational age intrauterine fetal growth (PE-AGAf). Controls were women with a physiologic pregnancy matched to cases on the basis of age (±6 months, ratio 2:1). Mitochondrial DNA copy number was quantified using real-time polymerase chain reaction and normalized to nuclear DNA. The median (interquartile range) mtDNAcn in peripheral blood in patients with PE-IUGR (n = 12) and in patients with PE-AGAf (n = 16) was 70 (44-97) and 108 (95-145), respectively (P = .004). Both these values were significantly lower than that detected in the control group (161[133-183], P < .001). The area under the receiver-operator curve for PE-IUGR and PE-AGAf were 0.94 (95% confidence interval [CI]: 0.88-1.00, P < .001) and 0.81 (95%CI: 0.70-0.91, P < .001), respectively. In conclusion, MtDNAcn in peripheral blood resulted significantly lower both in patients affected by PE-IUGR and in those affected by PE-AGAf when compared to controls. The accuracy of this biomarker resulted particularly good in predicting PE-IUGR.

Accumulation of retinoid X receptor-alpha in uterine leiomyomas is associated with a delayed ligand-dependent proteasome-mediated degradation and an alteration of its transcriptional activity.

An alteration of the retinoid pathway can influence the development of uterine leiomyomas in animal models, and retinoids have shown efficacy in inhibiting the growth of this benign tumor both in vitro and in vivo. However, the underlying mechanisms and biological implications are unclear. The present study was based on the demonstration of an accumulation of full-length retinoid X receptor alpha (RXRalpha) in leiomyomas that was not associated with a modification of its gene expression. This accumulation was shown to increase the transcription of the RXR-responsive gene cellular retinoic acid binding protein II (CRABP-II) and to be linked to the cellular redistribution of the receptor and to its retarded degradation via the ubiquitin/proteasome pathway. Accordingly, treatment with a specific proteasome inhibitor but not with protease inhibitors strongly inhibited the degradation of full-length RXRalpha in cells deriving from both myometrium and leiomyoma, but the formation of RXRalpha/ubiquitin conjugates was differentially regulated between the two cell types. Moreover, full-length RXRalpha accumulated in leiomyomas was abnormally phosphorylated at serine/threonine residues relative to myometrial tissue. The ligand to RXRalpha, 9-cis-retinoic acid, induced the receptor breakdown in smooth muscle cells deriving from both normal and tumor tissue, whereas a MAPK-specific inhibitor was able to reduce RXRalpha levels only in leiomyoma cells. These results suggest that switching of the ubiquitin/proteasome-dependent degradation of RXRalpha by phosphorylation in leiomyomas may be responsible for the accumulation of the receptor and the consequent dysregulation of retinoic acid target genes. The ability of retinoids to modify this molecular alteration may be the rationale for their use in the treatment of leiomyomas.

Pathobiological expression of the brain-derived neurotrophic factor (BDNF) in cerebellar cortex of sudden fetal and infant death victims.

Brain-derived neurotrophic factor (BDNF), a neurotrophin of the central nervous system, is able to regulate neuronal differentiation and modulate synaptic plasticity, being particularly involved in the development of the cerebellar cortical structure. The main aim of this study was to delineate, by immunohistochemistry, the BDNF expression in human cerebellar cortex of victims of fetal and infant death. The study was performed on a total of 45 cases, aged between 25 gestational weeks and 6 postnatal months, including 29 victims of sudden fetal and infant death and 16 age-matched subjects who died of known causes (Controls). We observed, in sudden death groups compared with Controls, a significantly higher incidence of defective BDNF expression in granule layers of the cerebellar cortex, which was particularly evident in the posterior lobule, a region that participates in respiratory control. These results were related to maternal smoking, allowing to speculate that nicotine, in addition to the well-known damages, can exert adverse effects during cerebellar cortex development, in particular in hindering the BDNF expression in the posterior lobule. This implies modifications of synaptic transmission in the respiratory circuits, with obvious deleterious consequences on survival.

Protective effects of vitamin D3 on fimbrial cells exposed to catalytic iron damage.

BACKGROUND: Recently, vitamin D3 (1alpha, 25-dihydroxyvitamin D) has shown its capability to take part in many extraskeletal functions and its serum levels have been related to patient survival rate and malignancy of many types of neoplasms, including ovarian cancers. Catalytic iron is a free circulating form of iron that is able to generate reactive oxygen species and consequently to promote a number of cellular and tissutal dysfunctions including tumorigenesis. In fertile women an important source of catalytic iron is derived from retrograde menstruation. Epithelial secretory cells from fimbriae of fallopian tubes are greatly exposed to catalytic iron derived from menstrual reflux and so represent the site of origin for most serous ovarian cancers. The aim of this study was to assess whether vitamin D3 can play a role in counteracting catalytic iron-induced oxidative stress in cells from fimbriae of fallopian tubes. METHODS: The cells, isolated from women undergoing isteroannessiectomy, were treated with catalytic iron 50-75-100 mM and vitamin D3 at a concentration ranging from 0.01 to 10 nM to study cell viability, radical oxygen species production, p53, pan-Ras, Ki67 and c-Myc protein expressions through Western Blot, and immunocytochemistry or immunofluorescence analysis. RESULTS: The pre-treatment with vitamin D3 1 nM showed its beneficial effects that consists in a significant decrease in ROS production. In addition a novel finding is represented by the demonstration that pre-treatment with vitamin D3 is also able to significantly counteract tumoral biomarkers activation, such as p53, pan-Ras, Ki67 and c-Myc, and consequently the catalytic iron-induced cellular injury. CONCLUSIONS: This study demonstrates for the first time that vitamin D3 plays an important role in preventing catalytic iron-dependent oxidative stress in cultured fimbrial cells. These results support the hypothesis that vitamin D3 could counteract carcinogenic changes induced by catalytic iron.

Maternal and fetal HLA-G 14 bp gene polymorphism in pregnancy-induced hypertension, preeclampsia, intrauterine growth restricted and normal pregnancies.

OBJECTIVE: Trophoblast expression of Human Leukocyte Antigene-G (HLA-G) is essential for feto-maternal immune tolerance and successful placentation. There is contradicting evidence on the relationship between HLA-G polymorphisms and preeclampsia (PE), intrauterine growth restriction (IUGR) and pregnancy-induced hypertension (PIH). Here, we investigate the association between both maternal and fetal HLA-G 14 bp insertion/deletion polymorphism and obstetrical complications. METHODS: Clinical and genetic data of 282 women/fetuses (31 severe PE, 8 mild PE, 46 IUGR, 42 PIH and 155 controls) were analyzed both individually and jointly under a codominant, a dominant and a recessive model. RESULTS: HLA-G 14 bp polymorphism was not associated with obstetrical complications, considering the mother and fetus genotypes both jointly and individually. CONCLUSIONS: With this study we filled several gaps occurring in previous studies: we analyzed a very well-defined population of PE, PIH and IUGR pregnancies, considering both fetal and maternal HLA-G 14 bp polymorphism, individually and jointly. Our findings showed that fetal and maternal HLA-G 14 bp genotypes are not associated with increased risk for the development of obstetrical complications, suggesting that this polymorphism has no immuno-modulatory role in the development of PE, PIH or IUGR.

Overexpression of the Wnt5b gene in leiomyoma cells: implications for a role of the Wnt signaling pathway in the uterine benign tumor.

CONTEXT: Uterine leiomyomas are the most common tumors in the human female pelvis and the leading indication for pelvic surgery. The molecular causes of the disease remain unknown. OBJECTIVE: Using an oligonucleotide microarray-based hybridization analysis, we observed that a Wnt family member transcript, Wnt5b, was overexpressed in smooth muscle cells (SMC) derived from leiomyomas when compared with matched myometrial cells. Based on this finding and on previous observations, we have hypothesized that altered expression of specific Wnt family members might be involved in leiomyoma formation and/or growth. MAIN OUTCOME MEASURES: The expression patterns of two members of the Wnt pathway, Wnt5b and secreted frizzled related protein (sFRP)1, were evaluated in myometrial SMC (n = 22) and in leiomyoma cells (n = 27) by real-time quantitative PCR. In addition, regulation of expression of the two molecules was examined. RESULTS: Compared with myometrial SMC, cells derived from leiomyomas had significantly higher levels of both Wnt5b and sFRP1 transcripts. When the data were analyzed as a function of the phase of the menstrual cycle, no significant difference in sFRP1 mRNA levels could be detected, whereas levels of Wnt5b transcript were significantly higher in the secretory phase in myometrial cells. Treatment with 9-cis retinoic acid significantly inhibited Wnt5b expression in myometrial SMC but not in their leiomyoma counterparts. CONCLUSIONS: Specific Wnt signaling genes are overexpressed in leiomyoma cells. Moreover, in these cells, the regulation of Wnt5b expression by retinoids appears to be attenuated.

Androgen receptor gene cytosine, adenine, and guanine trinucleotide repeats in patients with endometriosis.

OBJECTIVE: A genetic variation in the androgen receptor (AR) has been associated with the risk of developing endometriosis. The AR gene is located on the X chromosome and contains a highly polymorphic trinucleotide repeat (cytosine, adenine, and guanine: CAG) in its first exon, whose length and methylation pattern affect both AR expression and function. Thus, we sought to further investigate the potential association between endometriosis and the AR-CAG polymorphism. METHODS: Genomic DNA was obtained from a consecutive series of 197 white Italian women of reproductive age who underwent laparoscopy for benign gynecologic pathologies. Molecular analysis of AR-CAG repeats was performed by polymerase chain reaction amplification and Genescan evaluation. The pattern of CAG repeat distribution was compared between subjects with and without endometriosis. RESULTS: Endometriosis was documented in 105 women (stage I-II in 33 women and stage III-IV in 72 women). We found no difference in the number of AR-CAG repeats between women with endometriosis and controls. The CAG repeat length ranged from eight to 27 (mean +/- standard deviation, 17.4 +/- 1.9) for endometriosis patients and from 11 to 27 (mean +/- standard deviation, 17.4 +/- 2) for controls. Moreover, no association was found between AR gene polymorphisms and the various clinical manifestations of the disease. CONCLUSION: We conclude that AR-CAG repeat length does not constitute an important factor for the genetic predisposition to endometriosis.