{'fraction_non_alphanumeric': 0.032334384858044164, 'fraction_numerical': 0.024369085173501576, 'mean_word_length': 5.082014388489209, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 3, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
SERUM COBALAMIN STATUS OF PREGNANT WOMEN SUFFERING FROM GESTATIONAL DIABETES MELLITUS

.. Objectives: Determining serum cobalamin levels in Pregnant Women suffering from Gestational Diabetes mellitus (GDM) presenting at our tertiary care hospital. Study Design: Case control study. Setting: Department of Gynecology and Medicine, SMBB Medical College Layari General Hospital Karachi. Period: January 2016 to April 2017. Material & Methods: Sample of 100 pregnant women in 2nd and 3rd trimester was selected into; 50 controls and 50 GDM cases through convenient sampling. GDM was defined as pregnant women with fasting blood sugar ≥100 mg/dL. 5 ml blood was collected; 3 ml put into EDTA tubes for complete blood counts and 2 ml for sera. Blood glucose was estimated by hexokinase method, HbA1c by colorimetric method and cobalamin by ECLIA assay method. SPSS software 21.0 (IBM, Inc USA) was used for data analysis using Student t-test and Chi-square test (P ≤ 0.05). Results: Age of control was 30.43±1.49 years and 29.95±1.27 years in cases. Gestational age was 33.67±2.69 weeks in controls and 34.75±2.53 weeks in cases. Control and cases shows serum cobalamin levels of 316.34± 113.77 pg/ml and 253.5±121.32 pg/ml respectively (P=0.009). Serum cobalamin deficiency was noted in 68% of cases and 40% of controls (P<0.05). Glycemic control was bad in majority of cases. Serum cobalamin shows inverse correlation with random blood glucose, fasting blood glucose and Glycated HbA1. Conclusion: We found low serum cobalamin levels in pregnant women suffering from gestational diabetes mellitus that showed inverse correlation with random and fasting blood glucose and glycemic control.


INTRODUCTION
Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance and hyperglycemia first time observed during pregnancy. 1 GDM complicates approximately 7% of pregnancies accounting for >0.2 million cases per annum. [1][2][3] Prevalence of GDM ranges from 1-14% of pregnancies. The variation depends on the population studied and diagnostic methodology of glucose testing. Previous studies [2][3][4] form South East Asia reported GDM prevalence range of 4.2% to 26%. However, a recent study showed GDM prevalence of 17.2% in pregnant female during second trimester. 5 GDM is a serious metabolic disorder for mother and fetus. In mothers, the long term risk of DM is increased. Also the risk of metabolic syndrome and cardiovascular disease is increased in GDM. Pregnant GDM female are at increased risk of urinary tract infections, sepsis, pregnancy toxemia, and polyhydramnios. Incidence of premature child birth and cesarean section is increased too. Many of the fetal complications have been reported such as macrosomia, intrauterine death, congenital malformations, and spontaneous abortion. 6 Risk of developing type 2 DM is 7 times increased in subsequent pregnancies. 7 Many risk factors have been implicated for developing GDM, such as multiparity, maternal age ≥ 25 years and BMI >30 Kg/m 2 before pregnancy. Female suffering from GDM is prone to developing frank DM in next pregnancies, and post partum period. Female with history of macrosomia (>4.0 kg) of fetus, positive family history of DM, Asian and African ethnicities are at high risk of developing GDM in subsequent pregnancies. [1][2][3][4][5][6][7] Vitamin cobalamin (vitamin B 12 ) is an essential nutrient for the cell growth, maturation and cell division along with folic acid. Vitamin cobalamin plays role in the red blood cell production, neuronal growth and myelination of brain. 1,5,8 Vitamin cobalamin deficiency is prevalent in the country. 9, 10 Dietary vitamin cobalamin deficiency or due to increased demands as during pregnancy, it manifests as grave medical symptoms and complications in the mother and fetus. Delayed cobalamin replacement presents with rapid appearance of symptoms within short time period. Vitamin cobalamin deficiency may be treated by high dose oral supplements or parenteral route. 11 Pregnant women are at high risk of vitamin cobalamin deficiency because of poor nutritional status and high cell turnover in the body. Rapidly growing fetus consumes more vitamin cobalamin from the maternal blood thus putting them on increased risk of this vitamin that may be deficient in the maternal diet. If dietary requirements are not met, vitamin cobalamin deficiency ensues. 12 Vitamin cobalamin deficiency manifests as glossitis, stomatitis, red buffy tongue, and malabsorption due to villi atrophy. Mucositis is also noted. Peripheral blood smear shows macrocytic erythrocytes with or without anemia. Poikilocytosis, anisocytosis, ovalocytosis, hypersegmented neutrophils and pancytopenia may occur. 12,13 Fetus may develop intrauterine growth retardation, fatty liver disease and poor mental development due to defective myelination in the central nervous system. Such babies are prone to developing a multitude to chronic diseases in future such diabetes mellitus (DM), depression, cardiovascular disorders and even cancer. 12 Currently, the issue of vitamin cobalamin deficiency and GDM has been hotly hinted in medical literature 12,13 , hence there is a need to explore this association of Vitamin cobalamin and GDM in our indigenous population. The present study was planned to explore the Vitamin cobalamin status in Pregnant Women with Gestational Diabetes mellitus presenting at our tertiary care hospital.

MATERIAL & METHODS
The present case control study was conducted at the Department Gynecology and Obstetrics, Shaheed Muhtrama Benazir Bhutto Medical College Layari General Hospital Karachi, Sindh, Pakistan from January 2016 to April 2017. Ethical approval of research protocol was applied for the ERC (ethical review committee). Sample size was calculated by 'sampling for proportions'. Sample size was calculated to be 95 by using 5% α-level of significance and power of test of 90% at an expected % of vitamin cobalamin deficiency in GDM subjects as 51% and without GDM as 21.9%. 7 But in order to increase the power, 100 subjects were divided into 2 groups; Group 1cases of gestational diabetes mellitus and Group 2 -controls. Cases were selected by convenient sampling. GDM cases and control were selected according to the inclusion and exclusion criteria at the booked antenatal clinics of Layari General Hospital. GDM was defined according to the American Diabetes Association (ADA) criteria of 75g OGTT. 14 GDM was defined as pregnant women with fasting blood sugar ≥100 mg/dL. 14 Gestational age (GA) was calculated by recalling the first day of LMP (last menstrual period). Gravida was defined as total number of pregnancies regardless of the conception outcome. GDM cases were diagnosed as pregnant women with fasting blood sugar ≥100 mg/dL as cited. 14 A sample of 50 GDM patients were tagged cases. Similar number of age and gender matched pregnant female without diabetes mellitus were diagnosed as controls (n=50). Inclusion criteria for controls were pregnant female in 2 nd and 3 rd trimester of pregnancy with normal blood fasting sugar levels. Inclusion criteria for cases were defined as; 2 nd and 3 rd trimester of pregnancy of age 25 -40 years who remembered the exact 3 LMP date. And GDM patients being treated with metformin are strictly excluded. Pregnant female who suffered from abdominal tuberculosis, pancreatic disease, Gastroesophageal reflux disorder, strict vegetarians, renal disease, and chronic liver disease were excluded. Pregnant female using multivitamin pills since beginning of first trimester were excluded. Volunteers were informed about the purpose of study and willing volunteers were asked to sign the consent form. Findings of research interest were noted in a proforma of study protocol designed by the researcher. Patient history, gravidity, LMP, GA Clinical problem, and blood findings were noted. Confidentiality of patient's records was ensured. Patients were informed that the results and biodata will never be publicized and shall remain confidential. Volunteers were asked for blood sampling. Willing participants were informed that the blood sampling will cause no harm and blood will be used only for the testing of glucose and vitamin levels. Participants were intimated that the laboratory expense will be paid by the researcher not by the patients.
Height, weight and body mass index were calculated. Systemic blood pressure was measured by mercury sphygmomanometer. Willing volunteers were asked to lie on examination couch. A tourniquet was tightly applied above the cubital fossa to make the vein prominent. Skin over prominent vein was sterilized with alcohol swab. Disposable syringe (BD, USA) was used for venesection to draw 5 ml blood. 3 ml of blood sample was put into EDTA tubes for complete blood counts. Fasting blood sugar was estimated by glucose hexokinase method and hemoglobin A1 glycosylated (HbA1c) by Colorimetric method. Serum cobalamin levels were estimated by using the ECLIA (Electro-chemiluminescence immunoassay) method.
Serum cobalamin levels were defined as deficiency <200 pg/mL; marginal deficiency 200 -299 pg/mL and normal cobalamin at ≥300 pg/mL. 15 Data variables were typed on Excel sheet and copied to SPSS software 21.0 (IBM, Inc USA). Age, gestational age, gravida, fasting blood sugar, HbA1c and serum cobalamin were analyzed by Student`s t-test. Serum cobalamin categories of deficiency, marginal deficiency and normal were cross tabulated using Chi-square test. Pearson`s correlation was used for the correlation of serum cobalamin with RBG, FBG, HbA1c and gestational age. All data variables were analyzed at 95% confidence interval (P≤ 0.05).

RESULTS
Age of control was 30.43±1.49 years and 29.95±1.27 years of cases. Gestational age and gravida in control and cases were matched as shown in Table- Table-II). Sufficient serum cobalamin (≥300 pg/mL) was found in 30 (60%) controls compared to 16 (32%) GDM cases.
Marginal deficiency (cobalamin 200-299 pg/mL) was noted in 11 (22%) of cases compared to 15 (30%) of controls. 23 (46%) of cases revealed deficient serum cobalamin (<200 pg/mL) compared to 5 (10%) in controls. Total 68% cases and 40% controls revealed serum cobalamin deficiency (Table-III Table-IV. Figures-1-3 show the graphical distribution of negative correlation of serum cobalamin.  Table-  Serum cobalamin deficiency is prevalent 9,10 in the country due to nutritional deficiency added by economical constraints putting the pregnant ladies and children at highest risk.
Hence the finding of 40% cobalamin deficiency of present study is justified in the present scenario of nutritional deficiencies particularly in the developing countries. Many of previous studies [19][20][21] have attributed the serum cobalamin deficiency in diabetics to the metformin therapy but metformin intake was an exclusion criterion in the present study. Negative correlation of serum cobalamin with RBG, FBG, and HbA1c is important finding that show the glycemic control will be deviated more to abnormal side with concomitant cobalamin deficiency in pregnant women suffering from GDM. Serum cobalamin levels should be closely monitored for prevention of materno-fetal complications. Intrauterine life of fetal metabolic programming may adversely affect through epigenetic modifications making the fetus prone to chronic disease in adult life. 22,23 Timely correction of cobalamin deficiency and diabetic management of GDM may prevent the adverse feto-maternal outcomes. As the bad glycemic control is also a risk factor for the materno-fetal outcome hence this needs to be addressed by the treating obstetricians and physicians. The findings of present study in the light of national and international literature are worth to report. The limitations of present study include -first; small sample size, second; anthropometric data of pre-pregnancy serum cobalamin, nutritional status and dietary habits are not clear, third; cross sectional study design, hence findings cannot be generalized. However, the strength of study lays in its prospective study design, and exclusion and inclusion criteria.

CONCLUSION
We found low serum cobalamin levels in pregnant women suffering from gestational diabetes mellitus and showed inverse correlation with random and fasting blood glucose and glycemic control. The present hospital based study reports 68% deficiency of serum cobalamin in gestational diabetes mellitus. Cobalamin supplements should be recommended to this particular population at risk of improving maternal, neonatal and child outcome, giving emphasis on neurological processes and epigenetic modification. Copyright© 05 Nov, 2019.