Lavanya Rai

Erythrocyte indicators of oxidative stress in gestational diabetes

Foetuses born to mothers with gestational diabetes are at increased risk of developing respiratory distress, foetal macrosomia, foetal anomalies and platelet hyperaggregability. High blood glucose level induces oxidative stress and decreases antioxidant defences. The present study discusses the possibility of lipid peroxidation and protein oxidation in both maternal and foetal erythrocytes as an indicator of oxygen radical activity. The level of lipid peroxidation and protein oxidation in erythrocytes was estimated in 20 mothers with gestational diabetes and their newborns. The maternal age varied between 19 and 42 y and foetal age ranged between 34 and 39 weeks. The proteolytic activities in the erythrocyte lysates obtained from mothers with gestational diabetes and their newborns were significantly greater [(mean ± SD) 24.41 ± 9.05 and 16.70 ± 3.36μM of amino groups/g haemoglobin, n= 20, respectively] than those from control group (10.18 ± 4.84 and 14.64 ± 6.21 μM amino groups/g haemoglobin, n= 15, respectively; p < 0:05 in both cases). Similarly erythrocyte malondialdehyde levels were significantly elevated in babies born to mothers with gestational diabetes (10.11 ±2.21 nM/g haemoglobin) when compared to controls (6.8 ± 3.75 nM/g haemoglobin) (p < 0:05). In the erythrocytes of mothers with gestational diabetes, malondialdehyde levels correlated significantly with glycated haemoglobin levels (p < 0:01). The results of this study indicate that the oxidative stress induced by gestational diabetes manifests as increased lipid peroxidation and protein oxidative damage in the erythrocytes of both mothers with gestational diabetes and their newborn infants.

Optical diagnosis of cervical cancer by fluorescence spectroscopy technique.

In the present work, we examine normal and malignant stage IIIB cervical tissue by laser induced fluorescence, with 2 different objectives. (i) Development of the fluorescence spectroscopy technique as a standard optical method for discrimination of normal and malignant tissue samples and, (ii) Optimization of the technique by the method of matching of a sample spectrum with calibration sets of spectra of pathologically certified samples. Laser‐induced fluorescence spectra were measured using samples from 62 subjects at different excitation wavelengths. Principal component analysis (PCA) of spectra and intensity ratios of curve‐resolved fluorescence peaks were tested for discrimination. It was found that PCA of total fluorescence at 325 nm excitation gives specificity and sensitivity over 95%. Use of calibration sets of spectra of histo‐pathologically certified samples combined with PCA for matching and pass/fail classification of test samples is shown to have high sensitivity/specificity for routine diagnostic purposes as well as for possible staging of the disease. Further, the multi‐component origin of the fluorescence spectra is illustrated by curve resolution and fluorescence spectra of separated proteins of tissue homogenates.

METFORMIN — A CONVENIENT ALTERNATIVE TO INSULIN FOR INDIAN WOMEN WITH DIABETES IN PREGNANCY

OBJECTIVE To compare the use of metformin with that of insulin for the treatment of gestational diabetes mellitus (GDM) and type 2 diabetes mellitus (T2DM) unresponsive to diet therapy. MATERIALS AND METHODS In this prospective observational study, maternal glycemic control and perinatal outcome in diabetic pregnancies were compared between 2 obstetric units, one using insulin therapy and the other using metformin therapy. Baseline pretreatment glycemic profile was done and then repeated weekly throughout pregnancy. The outcome measures studied were glycemic control, maternal complications and perinatal outcome. RESULTS Sixty women with gestational and type 2 diabetes were enrolled, 30 each for metformin and insulin. Both groups were comparable with respect to age, body mass index (BMI), parity and pretreatment plasma glucose levels. Glycemic control was better with metformin after 1 week of therapy and also throughout gestation (P = 0.03-0.007). There were no major complications or perinatal deaths in this study. Mean gestational age and birth weight (2.9 +/- 0.4 kg versus 3.1 +/- 0.4 kg, P = 0.30) were comparable. However, there was a significant increase in neonatal intensive care unit (NICU) admission and stay for babies born in the insulin group. The cost of treatment was tenfold higher in thethe insulin group. CONCLUSION Metformin is clinically effective, cheap and a safe alternative to insulin therapy in pregnant diabetic women.

“Near Miss” Obstetric Events and Maternal Deaths in a Tertiary Care Hospital: An Audit

Objectives. (1) To determine the frequency of maternal near miss, maternal near miss incidence ratio (MNMR), maternal near miss to mortality ratio and mortality index. (2) To compare the nature of near miss events with that of maternal mortality. (3) To see the trend of near miss events. Design. Audit. Setting. Kasturba Hospital, Manipal University, Manipal, India. Population. Near miss cases & maternal deaths. Methods. Cases were defined based on WHO criteria 2009. Main Outcome Measures. Severe acute maternal morbidity and maternal deaths. Results. There were 7390 deliveries and 131 “near miss” cases during the study period. The Maternal near miss incidence ratio was 17.8/1000 live births, maternal near miss to mortality ratio was 5.6 : 1, and mortality index was 14.9%. A total of 126 cases were referred, while 5 cases were booked at our hospital. Hemorrhage was the leading cause (44.2%), followed by hypertensive disorders (23.6%) and sepsis (16.3%). Maternal mortality ratio (MMR) was 313/100000 live births. Conclusion. Hemorrhage and hypertensive disorders are the leading causes of near miss events. New-onset viral infections have emerged as the leading cause of maternal mortality. As near miss analysis indicates the quality of health care, it is worth presenting in national indices.

Maternal and fetal indicators of oxidative stress during intrauterine growth retardation (IUGR)

The present study demonstrates the possibility of increased lipid peroxidation and protein oxidation in both maternal and fetal erythrocytes as markers of oxygen radical activity during intrauterine growth retardation. The erythrocyte MDA levels were significantly elevated in mothers of IUGR babies when compared to controls (p<0.01). The endogenous protein damage due to oxidative stress was significantly higher in IUGR mothers when compared to controls (p<0.05). Similarly the proteolytic activity in erythrocyte lysates against oxidatively damaged hemoglobin was significantly increased in mothers of IUGR babies compared to controls (p<0.001).In fetuses born with IUGR, both lipid peroxidation and proteolytic activity were significantly increased when compared to normal newborns (p<0.01).The result of this study indicates that oxidative stress was induced both in IUGR babies and their mothers which is manifested as increased lipid peroxidation and protein oxidant damage.

DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain β (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer

Background: DOC2B promoter hypermethylation is an early and frequent event in cervical cancer. Results: DOC2B hypermethylation induces transcriptional repression, reactivated by demethylation; ectopic expression increases Ca2+ flux and inhibits key characteristics of tumorigenesis including proliferation, motility, and invasion. Conclusion: DOC2B gene is epigenetically regulated and inhibits cervical cancer growth. Significance: DNA methylation regulates DOC2B gene expression in cervical cancer. Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region −630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

Serum protein profile study of normal and cervical cancer subjects by high performance liquid chromatography with laser-induced fluorescence

High performance liquid chromatography with high sensitivity laser-induced fluorescence detection is used to study the protein profiles of serum samples from healthy volunteers and cervical cancer subjects. The protein profiles are subjected to principal component analysis (PCA). PCA shows that the large number of chromatograms of a given class of serum samples--say normal/malignant--can be expressed in terms of a small number of factors (principal components). Three parameters--scores of the factors, squared residuals, and Mahalanobis distance--are derived from PCA. The parameters are observed to have a narrow range for protein profiles of standard calibration sets formed from groups of clinically confirmed normal/malignant classes. Limit tests using match/no match of the parameters of any test sample with parameters derived for the standard calibration sets give very good discrimination between malignant and normal samples with high sensitivity (approximately 100%) aand specificity (approximately 94%).

Protein Profile Analysis of Cellular Samples from the Cervix for the Objective Diagnosis of Cervical Cancer using HPLC-LIF

Protein profiles of cytologic samples from the cervix were studied using High Performance Liquid Chromatographic (HPLC) separation combined with ultra-sensitive laser induced fluorescence (LIF) detection. HPLC-LIF protein profiles of samples from clinically normal subjects, individuals suffering from cervical cancer (different stages), and subjects who had other gynecological problems related to cervix, like erosion of cervix and Nabothian cyst, but no malignancy, were subjected to Principal Component Analysis (PCA). The application of HPLC-LIF protein profiling combined with PCA was found to be a highly efficient method for discrimination of different classes of samples with high sensitivity and specificity. Diagnostic accuracy and optimal threshold - decision criterion - for objective discrimination were estimated using sensitivity-specificity pairs and Youdens index (J) plots.

Protein profile study of the cervical cancer using HPLC-LIF

Optical methods and proteomics investigations are becoming promising approaches for early detection of many diseases, which remain clinically silent for long periods. We have used efficient High Performance Liquid Chromatography (HPLC) separation combined with highly sensitive laser induced fluorescence detection of proteins present in clinical samples for diagnostic applications in cervical cancer. The protein profile and the fluorescence of individual proteins were simultaneously recorded using our HPLC-LIF system. Protein profiles (Chromatogram) of serum from normal male and female volunteers with and without tobacco habits, and malignant serum samples were studied. Protein profiles were also recorded for lysates of exfoliated cells collected from Pap smear of normal and cancer patients. The protein profile patterns were subjected to Principal component Analysis. Discrimination of normal and malignant samples were achieved with very high sensitivity and specificity.

Significance of Maternal and Cord Blood Nucleated Red Blood Cell Count in Pregnancies Complicated by Preeclampsia

Objectives. To evaluate the effect of preeclampsia on the cord blood and maternal NRBC count and to correlate NRBC count and neonatal outcome in preeclampsia and control groups. Study Design. This is a prospective case control observational study. Patients and Methods. Maternal and cord blood NRBC counts were studied in 50 preeclamptic women and 50 healthy pregnant women. Using automated cell counter total leucocyte count was obtained and peripheral smear was prepared to obtain NRBC count. Corrected WBC count and NRBC count/100 leucocytes in maternal venous blood and in cord blood were compared between the 2 groups. Results. No significant differences were found in corrected WBC count in maternal and cord blood in cases and controls. Significant differences were found in mean cord blood NRBC count in preeclampsia and control groups (40.0 ± 85.1 and 5.9 ± 6.3, P = 0.006). The mean maternal NRBC count in two groups was 2.4 ± 9.0 and 0.8 ± 1.5, respectively (P = 0.214). Cord blood NRBC count cut off value ≤13 could rule out adverse neonatal outcome with a sensitivity of 63% and specificity of 89%. Conclusion. Cord blood NRBC are significantly raised in preeclampsia. Neonates with elevated cord blood NRBC counts are more likely to have IUGR, low birth weight, neonatal ICU admission, respiratory distress syndrome, and assisted ventilation. Below the count of 13/100 leucocytes, adverse neonatal outcome is quite less likely.