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.

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.

Length but not transverse diameter of the excision specimen for high-grade cervical intraepithelial neoplasia (CIN 2-3) is a predictor of pregnancy outcome.

The objective of this study was to analyze the impact of cone characteristics (depth, transverse diameter, and volume) on subsequent pregnancies after the loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia (CIN 2-3). Pregnancy outcomes (preterm birth, gestational age at birth, mode of delivery, and birth weight) of 501 women with singleton gestations and no previous preterm birth or history of late miscarriage, who had previously undergone a single LEEP for CIN 2-3, were retrospectively analyzed with respect to length, transverse diameter, and volume of the excision specimen. The overall incidence of preterm birth was 2.4%. The rate of preterm birth in women with length greater than 20 mm or volume greater than 2.5 cm was significantly higher than that in women with length between 15 and 19 mm (15.6 vs. 3.9%, P=0.02) or women with volume between 2.0 and 2.4 cm (5.8 vs. 1.6%, P=0.04). A linear inverse correlation (r=-0.3, P<0.001) between gestational age at birth and length, but not volume (r=0.0, P=0.9) or transverse diameter (r=0.2, P<0.0001), emerged. The mode of delivery was not affected by cone characteristics. Length, but not transverse diameter and volume, of the excised specimen seems to be related to a lower gestational age at birth. When excisions are performed under strict colposcopic guidance, with a correct modulation of cone length, the risk for preterm birth and cesarean delivery in subsequent pregnancies is not increased.

Vulvar lichen sclerosus: A new regenerative approach through fat grafting.

OBJECTIVE. The aim of this study was to evaluate the efficacy of fat grafting in the treatment of severe vulvar lichen sclerosus (LS). Our primary outcome was to assess the improvement of mucocutaneous trophism, the resolution/reduction of symptoms, and the histological features of the vulvar skin after treatment. The secondary outcome was to evaluate the improvement in life quality, and in resumption and quality of sexual life. METHODS. Between 2011 and 2014, 36 patients were offered fat grafting to treat LS. Inclusion criteria were age between 25 and 80 years, histopathologic diagnosis of LS, good health, failure of previous first line treatments. RESULTS. 34 out of 36 patients (94%) showed a better vulvar trophismof the skin and mucosae; 27 (75%) had an improvement in caliber and elasticity of the vaginal introitus; clitoris burying degree was reduced in 18 patients (50%), 30 (83%) reported an increased volume of labia major a and minor a, 34 (94%) had a complete disappearance of scratching lesions, and 28 (78%) showed a remission of white lesions. Eventually 34 patients (95%) stopped using topical corticosteroids routinely. The improvement in life quality was significant for both DLQI (p b 0001) and FSFI (p b 0001). CONCLUSIONS. Fat grafting may have a role as a support and completion treatment in selected cases of women with vulvar LS who do not respond to first line therapy or in severe cases where the anatomical impairment does not allow a regular sexual function and a good quality of life.

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.

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.

Depleted mitochondrial DNA content in peripheral blood of women with a history of HELLP syndrome.

OBJECTIVE: To test the hypothesis that a quantitative defect of maternal cellular mitochondria would play a role in the pathogenesis of HELLP syndrome. STUDY DESIGN: Peripheral blood mitochondrial DNA (MtDNA) was measured in 20 non-pregnant women with a history of HELLP syndrome, 40 non-pregnant control subjects who had previous physiologic pregnancies, 59 subjects carrying physiologic pregnancies, seven pregnant women with a history of HELLP syndrome and five women in the active phase of the disease. MAIN OUTCOME MEASURE: Peripheral blood Mt-DNA. RESULTS: The median (interquartile range) mtDNA in women with a history of HELLP syndrome, in non-pregnant women who had previous physiologic pregnancies, in subjects carrying physiologic pregnancies, in pregnant women with a history of HELLP syndrome and in women in the active phase of the disease was 115 (81-194), 229 (199-319), 174 (136-211), 101 (82-178) and 92 (39-129) copies per nuclear DNA, respectively. Non-pregnant women with a history of HELLP syndrome had significantly lower levels than non-pregnant controls (p<0.001). Moreover, blood mtDNA was lower in pregnant women with a history of HELLP syndrome and in those in the active phase of the disease when compared to pregnant controls (p=0.002 and p=0.025, respectively). CONCLUSIONS: Attenuated maternal mitochondrial function may favor HELLP syndrome development.

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.

Clear cell and papillary serous endometrial carcinomas: survival in a series of 128 cases.

PURPOSE: Clear cell (CC) and papillary serous carcinoma (PS) are histotypes at high risk of recurrence. We analyse patients' survival in a retrospective series of 128 CC and PS endometrial cancer cases. METHODS: All women with a histologically confirmed CC and PS endometrial cancer who underwent primary surgery in five institutions in Lombardy, Italy, were eligible for this study. A total of 77 (60.2 %) were PS endometrial cancer cases, 45 (35.2 %) CC cases and 6 (4.6 %) cases had mixed CC and PS histotype. RESULTS: 54 (42 %) cases were diagnosed at stage I, 10 (8 %) at stage II, 47 (37 %) at stage III and 17 (13 %) at stage IV. Recurrence was observed in 49 cases (38.3 %). The median time at recurrence was 12 months (interquartile range 7-18). The rate of recurrence was 20.3 % in cases at stage I-lI and 56.2 % in cases at stage III-IV (p < 0.0001). With regard to the site of recurrence 24 recurrences were in and 52 outside the pelvis. Finally, the rate of recurrence was 32.6 % (14 cases) in CC cases, 43.1 % (31 cases) in PS cases and 66.7 % (4 cases) in cases with mixed histotype. The 5-year progression-free survival was 59.5 % (67.4 % for CC cases, 55.1 % for PS and mixed cases). CONCLUSION: In this study including CC and PS endometrial cancers, the 5-year survival from surgery was 72.7 % and the 5-year progression-free survival was 59.5 %.

Compressive suture of the lower uterine segment for the treatment of postpartum hemorrhage due to complete placenta previa: a preliminary study.

AIM: To assess the efficacy of a uterine compression suture technique in controlling hemorrhage after removal of complete placenta previa during cesarean section. METHODS: This prospective study was performed in a tertiary referral hospital and included 18 patients with postpartum hemorrhage following removal of complete placenta previa during elective cesarean section. All 18 patients underwent bilateral anteroposterior compression suture of the lower uterine segment. All patients were followed postpartum for evaluation of uterine cavity and menstrual cycles. RESULTS: Anteroposterior compressive suture of the lower uterine segment achieved immediate complete hemostasis in all 18 patients. No surgical complication was observed. All patients recovered normal menstrual cycles. Normal patency of the uterine cavity was documented with sonohysterography in all patients at the 6-month follow-up visit. CONCLUSIONS: This quick and simple suture technique seems to be effective in stopping hemorrhage following complete placenta previa removal during cesarean section. Normal patency of the uterine cavity seems not to be impaired at medium-term follow-up.

The 'incessant menstruation' hypothesis: a mechanistic ovarian cancer model with implications for prevention.

Serous, endometrioid, clear cell and mucinous histotypes are the most common epithelial ovarian cancer. Most serous cancers appear to originate from precursor lesions at the fimbriated tubal end, whereas most endometrioid and clear cell cancers seem to derive from atypical endometriosis. Data regarding hormonal factors and associated gynaecologic conditions were critically analysed with the objective of defining a carcinogenic model for sporadic epithelial ovarian cancer complying with epidemiologic and pathologic findings. Oral contraceptives and tubal ligation substantially reduce the risk of serous, endometrioid and clear cell subgroups, but have no significant effect on mucinous tumours, which probably follow a different oncogenic pathway. We hypothesize that serous, endometrioid and clear cell cancers share a common pathogenic mechanism, i.e. iron-induced oxidative stress derived from retrograde menstruation. Fimbriae floating in bloody peritoneal fluid are exposed to the action of catalytic iron and to the genotoxic effect of reactive oxygen species, generated from haemolysis of erythrocytes by pelvic macrophages. This would explain the distal site of tubal intraepithelial neoplasia. Collection of blood inside endometriomas would lead to the same type of genotoxic insult on gonadal endometrial implants. This would explain why endometriosis-associated cancers develop much more frequently in the ovary than at extragonadal sites. In women not seeking conception, bilateral salpingectomy could be advised whenever planning surgery for independent indications, thus possibly reducing cancer risk, while preserving ovarian function. The use of oral contraceptives should be favoured for prolonged periods of time, especially in women with endometriosis, a population at doubled risk of gonadal malignancy.

Paclitaxel/carboplatin versus topotecan/paclitaxel/carboplatin in patients with FIGO suboptimally resected stage III-IV epithelial ovarian cancer a multicenter, randomized study.

OBJECTIVE: The objective of this prospective randomized phase III trial was to compare paclitaxel plus carboplatin (PC) versus topotecan plus carboplatin and paclitaxel (TPC) in women with suboptimal stage III (residual tumour >1cm) or stage IV ovarian cancer to evaluate the survival rate and toxicities. METHODS: Eligible for the study were patients aged at least 18 years old with histological/cytological diagnosis of FIGO stages III (residual tumour ≥1 cm after primary surgery)--IV epithelial ovarian cancer. Patients were randomized to iv PC on day 1, every 21 days or iv topotecan daily for three days and PC on day 3, every 21 days. RESULTS: The intention to treat population was made of 326 patients in total, 170 in the PC group and 156 in the TPC group. The life table estimates of survival probabilities at one, three and five years were, respectively, 0.94 (95% CI: 0.88-0.97), 0.53 (95% CI: 0.44-0.62) and 0.32 (95%CI: 0.23-0.42) in the PC group, and 0.92 (95% CI: 0.86-0.95), 0.52 (95% CI: 0.42-0.61), and 0.32(95%CI: 0.22-0.43) in the TPC group (log-rank test at 5 years: ns). The results of the survival analysis based on Cox regression model showed no statistically significant differences between groups (p-value: ns). The number of subjects with at least one event with possible relationship to study medication was 151 (88.8%) in the PC group and 139 (89.1%) in the TPC group (p=ns). In the PC group, 79 patients (23.6%) experienced at least one Adverse Event (AE) graded as severe and 16 patients (4.8%) at least one life-threatening AE, whilst in the TPC group, the number of patients who presented at least one severe or life-threatening AE was 86 (24%) and 37 (10.3%), respectively. CONCLUSION: The results of the present study show that the addition of topotecan to a standard paclitaxel/carboplatin regimen in the treatment of advanced epithelial ovarian cancer did not result in significant advantages in terms of survival rate. A slightly worse toxicity profile for TPC was observed.

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.

A clinical outcome-based prospective study on venous thromboembolism after cancer surgery: the @RISTOS project.

SUMMARY BACKGROUND DATA: The epidemiology of venous thromboembolism (VTE) after cancer surgery is based on clinical trials on VTE prophylaxis that used venography to screen deep vein thrombosis (DVT). However, the clinical relevance of asymptomatic venography-detected DVT is unclear, and the population of these clinical trials is not necessarily representative of the overall cancer surgery population. OBJECTIVE: The aim of this study was to evaluate the incidence of clinically overt VTE in a wide spectrum of consecutive patients undergoing surgery for cancer and to identify risk factors for VTE. METHODS: @RISTOS was a prospective observational study in patients undergoing general, urologic, or gynecologic surgery. Patients were assessed for clinically overt VTE occurring up to 30 +/- 5 days after surgery or more if the hospital stay was longer than 35 days. All outcome events were evaluated by an independent Adjudication Committee. RESULTS: A total of 2373 patients were included in the study: 1238 (52%) undergoing general, 685 (29%) urologic, and 450 (19%) gynecologic surgery. In-hospital prophylaxis was given in 81.6% and postdischarge prophylaxis in 30.7% of the patients. Fifty patients (2.1%) were adjudicated as affected by clinically overt VTE (DVT, 0.42%; nonfatal pulmonary embolism, 0.88%; death 0.80%). The incidence of VTE was 2.83% in general surgery, 2.0% in gynecologic surgery, and 0.87% in urologic surgery. Forty percent of the events occurred later than 21 days from surgery. The overall death rate was 1.72%; in 46.3% of the cases, death was caused by VTE. In a multivariable analysis, 5 risk factors were identified: age above 60 years (2.63, 95% confidence interval, 1.21-5.71), previous VTE (5.98, 2.13-16.80), advanced cancer (2.68, 1.37-5.24), anesthesia lasting more than 2 hours (4.50, 1.06-19.04), and bed rest longer than 3 days (4.37, 2.45-7.78). CONCLUSIONS: VTE remains a common complication of cancer surgery, with a remarkable proportion of events occurring late after surgery. In patients undergoing cancer surgery, VTE is the most common cause of death at 30 days after surgery.

Survival and prognostic factors of women with advanced ovarian cancer and complete response after a carboplatin-paclitaxel chemotherapy.

OBJECTIVE: We evaluated the characteristics and determinants of 5-year survival in ovarian cancer patients with complete response after first line treatment who entered a randomised study comparing two different chemotherapeutic schedules. METHODS: This analysis included 232 ovarian cancer patients with complete response after first line surgery and chemotherapy coming from a large randomised trial comparing the effect of different doses of paclitaxel combined with fixed doses of carboplatin. RESULTS: The 5-year overall survival in patients was 57.3%. The difference in 5-year survival for age <52 years (65.1%), 53-62 (51.4%) and > or = 63 (51.2%) was statistically significant (P = 0.048). The 5-year overall survival rates were 64.6% for stage III and 57.9% for stage IV. Serous and clear cell histotypes had a worse 5-year overall survival (51.5% and 50.8% respectively), while the endometrioid and mucinous had 67.1% and 71.4%: these differences were statistically different (P = 0.04). Women with residual tumour of 1 cm or smaller after primary surgery had better 5-year survival rates: 71.2% for patients with residual tumour < or = 1 cm and 46.9% for residual tumour >1 cm: these differences were statistically significant (P < 0.006). CONCLUSION: This study shows that in women with ovarian cancer and complete response after first line surgery and chemotherapy, age, histotype and residual tumour after surgery are determinants of 5-year overall survival.