An improved method for isolation of RNA from rat femur.

BACKGROUND: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. METHODOLOGY: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by ß-actin Ct values. RESULTS: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of ß actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). CONCLUSION: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR.

Transplantation Efficacy of Human Ciliary Epithelium Cells from Fetal Eye and Lin-ve Stem Cells from Umbilical Cord Blood in the Murine Retinal Degeneration Model of Laser Injury.

A number of degenerative conditions affecting the neural retina including age-related macular degeneration have no successful treatment, resulting in partial or complete vision loss. There are a number of stem cell replacement strategies for recovery of retinal damage using cells from variable sources. However, literature is still deficit in the comparison of efficacy of types of stem cells. The purpose of the study was to compare the therapeutic efficacy of undifferentiated cells, i.e., lineage negative stem cells (Lin-ve SC) with differentiated neurosphere derived from ciliary epithelium (CE) cells on retinal markers associated with laser-induced retinal injury. Laser-induced photocoagulation was carried out to disrupt Bruch's membrane and retinal pigmented epithelium in C57BL/6 mouse model. Lineage negative cells were isolated from human umbilical cord blood, whereas neurospheres were derived from CE of post-aborted human eyeballs. The cells were then transplanted into subretinal space to study their effect on injury. Markers of neurotropic factors, retina, apoptosis, and proliferation were analyzed after injury and transplantation. mRNA expression was also analyzed by real-time polymerase chain reaction at 1 week, and 3-month immunohistochemistry was evaluated at 1-week time point. CE cell transplantation showed enhanced differentiation of rods and retinal glial cells. However, Lin-ve cells exerted paracrine-dependent modulation of neurotrophic factors, which is possibly mediated by antiapoptotic and proliferative effects. In conclusion, CE transplantation showed superior regenerative outcome in comparison to Lin-ve SC for rescue of artificially injured rodent retinal cells. It is imperative that this source for transplantation may be extensively studied in various doses and additional retinal degeneration models for prospective clinical applications.

A study to compare maternal and perinatal outcome in early vs. late onset preeclampsia.

OBJECTIVE: The risk factors, clinical trends, and maternal and fetal health of early- and late-onset preeclampsia have not been adequately studied. We examined the effects of early- and late-onset preeclampsia on maternal and perinatal outcomes as well as the known risk factors of preeclampsia. METHODS: One hundred and fifty women with preeclampsia were consecutively enrolled in each group. Those who developed preeclampsia before 34 weeks of gestation were identified as having early-onset preeclampsia, while those who developed at 34 weeks or later were identified as having late-onset preeclampsia. Maternal and perinatal outcomes were compared between groups. RESULTS: Compared with the late-onset group, the early-onset group had higher rates of abruptio placentae (16% vs. 7.3%; P=0.019), but there was no intergroup difference in the composite maternal outcomes. A significantly higher number of women with early-onset preeclampsia developed severe features during the disease course, and most required treatment with antihypertensive drugs. Late-onset preeclampsia was more prevalent among primigravid mothers. Babies born to mothers with early-onset preeclampsia had a significantly higher rate of adverse outcomes. CONCLUSION: These study findings indicate that women with early-onset preeclampsia had more adverse outcome than those with late-onset preeclampsia, but the difference was not statistically significant. There were more babies with adverse perinatal outcomes in the early-than late-onset group.

Human Fetal Pigmented Ciliary Epithelium Stem Cells have Regenerative Capacity in the Murine Retinal Degeneration Model of Laser Injury.

BACKGROUND: Retinal degeneration and related eye disorders have limited treatment interventions. Since stem cell therapy has shown promising results, ciliary epithelium (CE) derived stem cells could be a better choice given the fact that cells from eye niche can better integrate with the degenerating retina, rewiring the synaptic damage. OBJECTIVE: To test the effect of human fetal pigmented ciliary epithelium-derived neurospheres in the mouse model of laser-induced retinal degeneration. METHODS: C57 male mice were subjected to retinal injury by Laser photocoagulation. Human fetal pigmented ciliary epithelium was obtained from post-aborted human eyeballs and cultured with epidermal growth factor (rhEGF) and fibroblast growth factor (rhFGF). The six day neurospheres were isolated, dissociated and transplanted into the subretinal space of the laser injured mice at the closest proximity to Laser shots. Mice were analyzed for functional vision through electroretinogram (ERG) and sacrificed at 1 week and 12 week time points. Retinal, Neurotropic, Apoptotic and proliferation markers were analysed using real-time polymerase chain reaction (PCR). RESULTS: The CE neurospheres showed an increase in the expression of candidate genes analyzed in the study at 1 week time point, which sustained for longer time point of 12 weeks. CONCLUSION: We showed the efficacy of human CE cells in the regeneration of retinal degeneration in murine model for the first time. CE cells need to be explored comprehensively both in disease and degeneration.

Conventional radiotherapy and intensity-modulated radiotherapy in carcinoma vulva: An experience from a tertiary medical center of India.

CONTEXT: Vulvar cancer is one of the uncommon gynecological malignancies. Multimodality treatment with surgery, radiotherapy, and chemotherapy are required for treatment of the disease. AIMS: The aim of the study was to evaluate clinical outcome in patients of carcinoma vulva, treated at our institution. SUBJECTS AND METHODS: This was a retrospective-cohort study done in 50 patients with squamous cell carcinoma of the vulva, treated at our institution from January 2008 to December 2014. Data were analyzed on the basis of age, stage, type of treatment received, and treatment-related toxicity. Disease-free survival and overall survival were estimated. STATISTICAL ANALYSIS USED: Kaplan-Meier survival analysis and Chi-square test were used for statistical analysis. RESULTS: Majority of the patients (52%) had presented with Stage III disease. Thirty-six of 50 patients underwent surgery: simple vulvectomy - 2, radical vulvectomy - 34, bilateral inguinal lymph node dissection was done in 32 patients, and 1 patient underwent ipsilateral-inguinal lymph node dissection. Among 40 patients who received radiotherapy and eight patients received palliative radiotherapy. Seventeen patients underwent intensity-modulated radiotherapy (IMRT) and 15 patients received conventional radiotherapy. Significantly less Grade 2 or more skin toxicity (P = 0.003) observed in patients who underwent IMRT. Among non-IMRT group, eight patients required treatment break during radiation. At a median follow-up time of 25.5 months, median overall survival was 31 months and median disease-free survival was 25 months. About 42% patients were alive and free of disease at last follow-up. CONCLUSIONS: Modified radical vulvectomy with inguinal lymph node dissection followed by radiotherapy is the mainstay of management of locally advanced carcinoma vulva. Using IMRT, we could minimize the treatment related radiation toxicity and treatment breaks.

Resveratrol restores the level of key inflammatory cytokines and RANKL/OPG ratio in the femur of rat osteoporosis model.

Resveratrol's effect on bone mineral density (BMD) and expression of cytokines in ovariectomized rats (postmenopausal osteoporosis model) was studied. The study was conducted on 3-month-old Sprague-Dawley rats that were (a) sham-operated, (b) ovariectomized, (c) ovariectomized and treated with ß-estradiol (487.5 µg/kg weight/day), and (d) ovariectomized and treated with resveratrol (625 µg/Kg body weight/day). The treatment was for 4 weeks. After sacrifice BMD and gene expression (RANKL, OPG, IL-23, and IL-17A, IL-1ß, and TNFα) were measured in tibia and femur respectively. Resveratrol could restore RANKL/OPG ratio, slightly increase BMD, and moderately but significantly reduce IL-23, IL-17A, IL-1ß, and TNF-α cytokine expression levels.

Effect of maintenance tocolysis with nifedipine in established preterm labour on pregnancy prolongation and neonatal outcome.

Fifty women with singleton pregnancies between 26+0/7 and 33+6/7 weeks of gestation and arrested preterm labour (PTL) after acute tocolysis were randomised by a computer generated randomisation table into an intervention group (n = 25) who received maintenance tocolysis with tablet nifedipine for 12 days or up to 34 weeks of gestation, whichever was later and a control group (n = 25). The primary outcome was achievement of term gestation and the secondary outcomes were the number of days gained till delivery and neonatal mortality and morbidity. The mean gestation at admission, cervical dilatation and effacement were similar in the two groups (30 + weeks, 2.5 cm, 60%). In the intervention group, 7/25 (28%) and in the control group, 2/25 (8%) delivered at term (p = .066) and pregnancy prolongation of 20 days (IQR 2.5-51) and 14 days (IQR 1-27.5) were achieved, respectively (p = .269). Maintenance tocolysis was given for a median of 14 days (range 3-25.5). Kaplan-Meier analysis showed no statistically significant difference in prolongation of pregnancy between the control and the intervention groups (p = .077). The median number of days of neonatal hospital stay were reduced with maintenance tocolysis, but the difference was not significant (4.0 vs 5.5; p = .608). The mean birth weight was significantly higher in the intervention group (2266 vs 1880 g, p = .044). Among women at a high risk for preterm birth (PTB) due to established PTL as evidenced by a mean cervical dilatation of 2.5 cm and a PTB rate of 92% in the control group, maintenance tocolysis did not prolong the pregnancy or reduce the neonatal hospital stay significantly. Impact statement What is already known on this subject: In women with preterm labour (PTL) the role of maintenance tocolysis following acute tocolysis to reduce recurrent PTL is uncertain. Of the six studies using nifedipine, one reported pregnancy prolongation (26.65 vs 16.14 days, p = .007), but similar perinatal outcome (Sayin et al. 2004 ). Others did not find pregnancy prolongation (Carr et al. 1999 ; Lyell et al. 2008 ; Uma et al. 2012 ; Roos et al. 2013 ; Parry et al. 2014 ). The PTB rate in the control groups ranged from 38 to 67%. A Cochrane review reported pregnancy prolongation by 5.35 days but similar neonatal outcome (RR 0.75) (Naik et al. 2013 ). A meta-analysis including five studies using progesterone and five using nifedipine concluded that progesterone, but not nifedipine, prolonged pregnancy (Ding et al. 2016 ). Thus, data on maintenance tocolysis is limited and inconclusive. What the results of this study add: In the present randomised study in 50 women with arrested PTL, 25 received maintenance tocolysis. The mean gestation at admission, cervical dilatation and effacement were similar in the two groups (30+ weeks, 2.5cm, 60%). In the intervention group, 7/25 (28%) and controls, 2/25 (8%) delivered at term (p = .066) and pregnancy prolongation of 20 days (IQR 2.5-51) and 14 days (IQR 1-27.5) were achieved, respectively (p = .269). Kaplan-Meier analysis showed no statistically significant difference in prolongation of pregnancy between the control and the intervention groups (p = .077). The median number of days of neonatal hospital stay were reduced with maintenance tocolysis but the difference was not significant (4.0 vs 5.5; p = .608). What are the implications of these findings for clinical practice and/or future research: The mean birth weight was higher in the intervention group (2266 vs 1880g, p = .044). Future studies should take cervical dilatation and the PTB rate in the control group into consideration while assessing the impact of maintenance tocolysis.

A Rare Case of Successfully Treated Coombs Negative Immune Haemolytic Anaemia in Pregnancy.

Immune haemolytic anaemia in pregnancy, although rare, but it can be life threatening. Severe anaemia with jaundice, unresponsive to blood transfusion can clinch the diagnosis of immune haemolytic anaemia. Our patient was a 27-year-old second gravida, with all the above features, but there was diagnostic challenge as her Coombs test was negative. A high index of suspicion and rapid response to glucocorticoids, pointed towards the diagnosis. Thereafter, the course of pregnancy and postpartum period was uneventful. Thus, successful maternal and fetal outcome can be achieved with prompt diagnosis and treatment.

Scrub typhus in pregnancy: a case series.

Scrub typhus, an acute febrile illness caused by Rickettsia and transmitted by mites, is a re-emerging endemic zoonosis in the Asia Pacific region. It is an uncommon entity and very few cases of this disease in pregnant women have been reported. We present a series of six such cases collected over 1 year with poor feto-maternal outcome in 50%.

Vascular endothelial growth factor (VEGF) induced proliferation of human fetal derived ciliary epithelium stem cells is mediated by jagged-N cadherin pathway.

The pigmented ciliary epithelium (PCE) of mammalian eye harbors resident population of stem cells that lie in apposition with endothelial cells which release vascular endothelial growth factor (VEGF) that may influence the fate and function of these stem cells in ways that remain unclear. We examined the role of VEGF in proliferation of PCE stem cells and expression of Notch, Jagged, N-Cadherin and ß-Catenin which are known to maintain proliferation state of neural stem cells. We cultured human PCE cells obtained from 12-20 weeks old fetal eyes. The neurospheres were analyzed for the proliferation capacity of PCE stem cells in presence of VEGF on 3,6 and 9 day. Real time PCR was used to quantitate the mRNA expression of above mentioned genes on PCE derived neurospheres on 3,6 and 9 day. We found increased number of neurospheres when PCE stem cells were stimulated with VEGF along with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) than EGF and bFGF. BrdU immunostaining was done to analyze the proliferation of CE cells and presence of neural and retinal progenitor markers such as Nestin and Pax6 were also investigated. An increased Notch and Jagged mRNA was observed on 6(th) day in VEGF, EGF and bFGF treated PCE cells as compared to 0,3 and 9 day. A similar pattern was noticed with N-cadherin and ß-catenin mRNA levels. These findings may clarify the role of VEGF on PCE stem cell proliferation with possible involvement of Notch, Jagged, N-cadherin and ß-Catenin. The data may suggest importance of harvesting 6(th) day neurospheres for transplantation purposes in preclinical investigations pertaining to retinal degenerative diseases, however, additional studies are needed to substantiate the findings.

Pelvic artery embolization in the management of obstetric hemorrhage.

We studied the role of pelvic artery embolization in management of obstetric hemorrhage by retrospective analysis of 50 cases of obstetric hemorrhage in a tertiary care referral hospital where this procedure was used. Uterine and or internal iliac artery embolization was performed for the management of postpartum hemorrhage (39 cases; 78%), post abortal bleeding (6 cases; 12%) and gestational trophoblastic disease (4 cases; 8%). In one case of postpartum hemorrhage procedure could not be performed due to arterial spasm (2%). Prophylactic embolization was carried out in one case of placenta accreta. The mean age of the women was 27 years and 54% were primiparas. In six women embolization was done after hysterectomy failed to control pelvic hemorrhage. One fourth of women had coagulopathy at the time of presentation. There were five cases of pelvic hematoma and three cases of arteriovenous malformations. The success rate of the procedure was 94% and the procedure was unsuccessful for controlling bleeding in three women. There were no major procedure related complications. Thus, pelvic artery embolization is an effective alternative to surgery in controlling obstetric hemorrhage and as a fertility and life-saving procedure.

Intrapartum antibiotics and risk factors for early onset sepsis.

OBJECTIVE: To determine the independent risk factors for early onset neonatal sepsis (EONS) in a setting where the policy is to use intrapartum antibiotic prophylaxis (IAP) for known risk factors. DESIGN: Prospective cohort study. SETTING: Level III neonatal unit in a developing country. PATIENTS: Consecutive mother-infant dyads (gestation < or =34 weeks) with no major neonatal malformations. INTERVENTIONS: Thirteen putative maternal and neonatal risk factors and use of IAP were assessed. Neonates were followed until 72 h of life for signs of EONS. Blood cultures were drawn on clinical suspicion of EONS and/or prior to starting prophylactic antibiotics for high risk asymptomatic neonates. MAIN OUTCOME: Culture-proven EONS (onset at <72 h). RESULTS: 601 mother-infant dyads were enrolled (mean (SD) gestation=31.8 (2) weeks; mean (SD) birth weight 1559.4 (452) g). The best fitted multivariable logistic regression model had six independent risk factors (adjusted OR (95% CI)): vaginal examinations > or =3 (9.5 (3 to 31)), clinical chorioamnionitis (8.8 (2 to 43)), birth weight <1500 g (2.8 (2 to 5)), male sex (2.7 (2 to 5)), gestation <30 weeks (2 (1 to 4)) and no IAP (2 (1.04 to 4)). Regression coefficients were converted into scores of 6, 6, 3, 3, 2 and 2, respectively. Internal prediction accuracy was 86.5% and c statistic was 0.75 (95% CI 0.70 to 0.81, p<0.001). CONCLUSIONS: Vaginal examinations > or =3, clinical chorioamnionitis, birth weight <1500 g, male sex, gestation <30 weeks and no intrapartum antibiotics were independent risk factors for EONS among preterm infants of < or =34 weeks' gestation.

Obstetric morbidity and the diagnostic dilemma in pregnancy in rudimentary horn: retrospective analysis.

BACKGROUND: Pregnancy in rudimentary horn of uterus, a form of ectopic gestation, is associated with significant rates of morbidity and mortality. Despite the recent advances in the ultrasonography, diagnosis of cornual pregnancy still remains elusive; with confirmatory diagnosis usually made during laparotomy. The aim of the present study is to analyze the obstetric implications and the diagnostic dilemma of rudimentary horn pregnancy. MATERIALS AND METHODS: Records of women diagnosed with ectopic pregnancy in the rudimentary horn, during the years 2004 to 2008, managed in a referral hospital in northern India; were reviewed for their diagnostic difficulties and the associated morbidity. RESULTS: During the four year study period, rudimentary horn pregnancies accounted for 12 pregnancies. Non communicating horn accounted for 75% of the cases. The mean age of women at presentation was 26 +/- 5.11 years and the period of gestation at diagnosis varied between 10 and 34 weeks. Preruputure diagnosis was possible only in two cases and sensitivity of ultrasonographic diagnosis was 33.3%. Laprotomy with excision of rudimentary horn and salpingectomy was done in all cases. Multiple blood transfusions were required in 83.3% of women. CONCLUSION: Management of pregnancy in a rudimentary uterine horn continues to be a challenge to this day. Maintaining a higher degree of alertness, especially in high risk groups by emergency staff is required to prevent the morbidity, as they may present with acute uterine rupture in pregnancy.

Congenital vesicovaginal fistula with transverse vaginal septum and ectopic ureter opening in proximal vagina: case report and brief review.

Congenital vesicovaginal fistula is an extreme rarity. We report on a case of a 22-year-old lady who presented with menouria and infertility. On evaluation, she was found to have congenital vesicovaginal fistula, a nonfunctioning right kidney with ectopic ureter and transverse vaginal septum. Abdominal repair of the fistula, right nephroureterectomy, and excision of the vaginal septum was performed.

Pheochromocytoma associated with pregnancy.

Pheochromocytoma associated with pregnancy is rare with potentially lethal consequences. Antepartum diagnosis improves the maternal and perinatal outcome. The issue of mode of delivery is unresolved. Its definitive treatment is surgical resection preceded by medical management. Surgical resection may be done during caesarean section as is reported in the present case.