RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, KARNATAKA,

BANGALORE.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE : DR. CHRISHANTHI QUIRINE RODRIGUES

ADDRESS : DEPARTMENT OF BIOCHEMISTRY

ST. JOHNSMEDICALCOLLEGE

SARJAPUR ROAD

BANGALORE– 560034.

2. NAME OF THE INSTITUTION: ST. JOHN’S MEDICAL COLLEGE

3. COURSE OF STUDY AND

SUBJECT : MD BIOCHEMISTRY

4. DATE OF ADMISSION TO

COURSE : 18-03-2009

5. TITLE OF THE TOPIC : A STUDY TO EVALUATE GLYCATED

HEMOGLOBIN LEVELS IN PREGNANCY

IN A TERTIARYCAREHOSPITAL.

6.BRIEF RESUME OF THE INTENDED WORK.

6.1: NEED FOR THE STUDY

Diabetes is a major cause of perinatal morbidity and mortality as well as maternal morbidity. In a study conducted in the year 2000, to estimate the global prevalence of Diabetes, India was found to have the largest number of diabetics(31.7 million) and by the year 2030 it is estimated to be about 79.4 million.1

Studies on pregnant women have shown that women with diabetes have undesirable pregnancy outcomes compared to non-diabetic mothers, the wide spectrum of complications being increased rates of obstetric complications, still births, perinatal mortality, congenital malformations, macrosomia and increased risk for pre-term delivery.2-5 These various maternal and foetal complications in diabetic pregnancies can be minimized by early detection of diabetes and strict glycemic control.6 A study in Indian population has also shown that tighter glycemic control can favourably alter adverse outcome parameters in case of Gestational Diabetes Mellitus patients.7

Studies done in the second trimester 8 and third trimester 9 of pregnancy have shown that poor maternal glycemic control is associated with neonatal morbidity. Improvement of obstetric and newborn care in these patients has resulted in a significant reduction in neonatal mortality and morbidity over the last few decades.10 However progress has been slow in some areas of clinical management especially in the Indian population. Hence there is a need to create awareness regarding the importance of tight glycemic control and also to know the usefulness of Glycated hemoglobin in this aspect. Glycated hemoglobin (HbA1c) is a reliable marker for the assessment of glycemic control.11 When measured at different intervals it can give an idea regarding the overall control before conception, during pregnancy and in the post partum period and thereby can help in diabetic management. However, it is relevant and practical to know the physiological levels of HbA1c during normal pregnancy before interpreting the results.

According to the American Diabetes Association, HbA1c levels should be within 1% above the upper limit of the normal range in adults in order to have good glycemic control and a rate of complications no greater than those in non-diabetic pregnancies.4 Inspite of following these guidelines and achieving good glycemic control, studies have shown that such levels of control are still not good enough for preventing the occurrence of perinatal complications.12

The current criterion for strict glycemic control is not safe enough and the targets need to be revised. Studies on HbA1c in normal pregnancy have shown mixed results, with some showing an increase, 13,14 others a decrease 15,16 and yet others no change.17, 18 Also there is paucity of literature regarding the levels of HbA1c in pregnancy.

Hence this study intends to evaluate the levels of Glycated hemoglobin in the second trimester of pregnancy and to compare these levels to the levels in the non-pregnant state.

6.2: REVIEW OF LITERATURE.

About one to twenty percent of the pregnancies in this world are found to be complicated by Diabetes. Gestational diabetes mellitus is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. Gestational diabetes mellitus represents nearly 90% of all pregnancies complicated by diabetes. However, the prevalence may range from 1-14% depending on the population studied.19

The mortality associated with diabetic pregnancies was very high in the early part of the 20th century. Maternal mortality in pregnancy drastically reduced to almost the general population level following the use of insulin in the treatment of diabetes and with improved glycemic control perinatal complications have also decreased.20

Over the past years several studies have been undertaken in different parts of the world to know the adverse outcomes of diabetes on pregnancy. The adverse outcomes were found to be more common among infants born to mothers with diabetes. A French multicentric survey showed about a six-fold increase in major congenital malformations and a eight-fold increase in pre-term delivery in diabetic pregnancies.3

Indian studies undertaken in this area have also shown similar outcomes. A study undertaken in St. JohnsMedicalCollege, Bangalore found respiratory distress syndrome, hypoglycemia, hyperbilirubinemia and macrosomia to be the most common perinatal problems in diabetic pregnancy.2 A recent study done in Chennai showed that women with diabetes have worse pregnancy outcomes compared to non-diabetic mothers and those with pre-gestational diabetes mellitus far worse than those with gestational diabetes mellitus.5

All these studies therefore emphasize on the fact that early detection of diabetes and strict glycemic control is very essential for the successful outcome of a pregnancy.

Glycated hemoglobin in the assessment of glycemic control.

HbA1c is formed by the condensation of glucose with the N-terminal Valine residue of each beta chain of HbA to form an unstable schiff base, which may either dissociate or undergo an Amadori rearrangement to form a suitable ketoamine, HbA1c. Formation of Glycated hemoglobin is irreversible and the concentration in the blood depends on both the lifespan of the RBC and the blood glucose concentration. Glycated hemoglobin concentration represents the integrated values for glucose over the preceding six to eight weeks.

In the 1960’s, Glycated hemoglobin was first identified in patients with diabetes. Later on it was thought that measurement of HbA1c could be useful as a measure of glucose control and hence studies correlating it to blood glucose levels were undertaken. It was introduced into widespread clinical use in the 1980’s.

Currently, it is recommended that all patients with diabetes should get their HbA1c levels measured in order to document their degree of glycemic control.11 It is a simple and economical way for assessment of long term glycemic control.

In a study done in France, congenital malformations of 4.4 %( twice the general population) were seen in women with HbA1c more than 8%. 3 In some other studies, despite the HbA1c levels being less than 7%, incidence of congenital malformations and macrosomia were found to be high.12 Thus it can be concluded that these levels of maternal glycemic control thought to be optimal are still not safe enough.

Glycated hemoglobin in pregnancy.

The use of any measurement in pregnancy should take account of pregnancy –specific normal ranges. Thus results of Glycated hemoglobin in pregnancy should be interpreted in the knowledge of these demonstrable physiological changes.

In a normal pregnancy, between six to ten weeks, there is a decrease in the fasting blood glucose and this continues throughout pregnancy and thereby the newer RBC’s are exposed to lower glucose concentration.21In addition to this, the RBC life span is also reduced in pregnancy. Hence, the HbA1c values in pregnancy can be expected to be lower than in the non-pregnant state.

The normal reference interval for HbA1c is well established in adults but not clearly defined in pregnancy. In a report by Worth et al, the HbA1c levels were found to be increased in pregnancy.13 A similar increase was also seen in another study done by Vintzileos AM et al.14On the other hand, a study conducted by Fadel HE et al showed no change in HbA1c levels in pregnancy.17 This was in good agreement with the study by O’Shaughnessy et al.18Furthermore, there are another set of studies, which show that HbA1c levels are lower in the pregnancy state. Nielsen et al demonstrated a decrease in the upper reference limit of HbA1c from 6.3% before pregnancy to 5.7% in early pregnancy and 5.6% in the third trimester.15Radder et al have also reported a fall in the HbA1c levels during pregnancy.16

From the above studies it is clear that the normal levels of HbA1c in pregnancy is still a controversial issue. An Indian study done to find levels of HbA1c in gestational diabetes mellitus has shown the mean A1c of women with normal glucose tolerance to be 5.3%. 22 There is paucity of Indian studies regarding the HbA1c levels in healthy pregnancy.

It is therefore important to conduct further studies in this area especially in the Indian population with an intention of achieving good glycemic control and thereby trying to minimize perinatal mortality and morbidity.

6.3: OBJECTIVES OF THE STUDY.

  1. To assess the HbA1c level in the second trimester of pregnancy.
  1. To compare the HbA1c levels in pregnant and the non-pregnant state.
  1. To correlate HbA1c values in the second trimester of pregnancy with Oral Glucose Challenge Test (OGCT) values.

7. MATERIALS AND METHODS.

7.1: Source of data:

(1) The study will be conducted on the pregnant women in the second trimester

visiting the Antenatal care clinic at the Obstetrics and Gynecology OPD of a

tertiary care hospital.

(2)Operational definitions

Cases:

The cases are defined as antenatal women in the age group of 18-35 years in their second trimester with a normal Oral glucose challenge test (OGCT).

Controls:

The controls are defined as non-pregnant healthy women in the age group of 18-35 years.

(3)Subjects who satisfy the inclusion criteria and give consent will be included in

the study.

(a)The number of subjects required was calculated using a study done by

Nielsen et al15. In this study, there was a difference of 0.3 in the mean HbA1c level between the pregnant and the non-pregnant women and the standard deviation was 0.3. The sample size needed to prove this difference was calculated with the help of n-Master software programme taking the power of the study to be 80% and alpha-error of 0.05% and was found to be 16. Hence the sample size taken is 20.

(b)For cases:

Inclusion criteria:

  1. Pregnant women in the second trimester.
  2. Age group of 18-35 years.

Exclusion criteria:

  1. Abnormal OGCT
  2. History of Diabetes Mellitus.
  3. Previous history of gestational diabetes mellitus, large babies, still births.
  4. History of smoking and alcohol consumption.
  5. Current consumption of any diabetogenic drugs.
  6. Iron deficiency anemia.
  7. Hemolytic anemia.

For cases:

Inclusion criteria:

  1. Women in the age group of 18-35 years (age matched).
  2. Non-pregnant.

Exclusion criteria:

  1. History of Diabetes Mellitus.
  2. Chronic steroid medication.
  3. Gestational diabetes mellitus in the past.
  4. Any significant hemoglobinopathies in the past.
  5. Iron deficiency anemia.
  6. Hemolytic anemia.

7.2: Method of collection of data.

7.2 a: Method of sample collection

Informed consent will be obtained from all the subjects included in the study.

The pregnant women in the second trimester attending the Antenatal care clinic will have to undergo a Oral Glucose Challenge Test (OGCT) with 50 grams of glucose. Blood samples will be collected in vacutainers by venepuncture with aseptic precautions. Only those with a normal OGCT will be taken for the study.

Blood samples for estimation of Glycated hemoglobin will be collected on the same day by venepuncture in EDTA anticoagulated vacutainers.

7.2 b: Sample size: 20 cases and 20 controls.

7.2 c: Type of study: Cross-sectional study.

7.2 d: Statistical analysis: Data collected will be given as means +/- SD.

Independent student t test will be used to compare the two groups.

7.3Does this study require any investigations or interventions to be conducted on patients or other human or animals? If so, please describe briefly.

The study requires the following investigations to be done on humans.

a)Oral Glucose Challenge Test.

A 50-gm oral glucose load is administered to the patient followed by a serum glucose determination at 1 hour. A value less than 140mg/dl is considered normal.

Serum glucose measurement by HK\G6PD method on Dade RxL analyser.

Hexokinase catalyses the phosphorylation of glucose in the presence of

adenosine –5-triphosphate and magnesium to form glucose 6-phosphate and adenosine di-phosphate. Glucose 6-phosphate is then oxidized by glucose 6 phosphate dehydrogenase in the presence of NAD to produce 6 phospho gluconate and NADH. 1 mole of NAD is reduced to 1 mole of NADH for each mole of glucose present. The absorbance due to NADH is determined using a bichromatic end point technique.

b)Blood Glycated hemoglobin

HbA1c measurement by HPLC method on BIORAD D-10TM analyzer.

Principle : The D-10 hemoglobin testing system chromatographically separates labile A1c and carbamylated hemoglobin from hemoglobin A1c. The BIORAD D-10 HbA1c analytical cartridge is used in conjunction with the HbA1c programme and is intended for the percent determination of HbA1c in human whole blood using ion exchange high performance liquid chromatography.

Blood samples will be collected from the subjects by venepuncture observing all safety and aseptic precautions. Blood samples for glucose estimation will be collected in red capped vacutainers and for Glycated hemoglobin in EDTA anti-coagulated vacutainers.

The patients will not be charged for the investigations done for the purpose of the

study.

7.4 Has ethical clearance been obtained from your institution?

Yes (consent form enclosed).

8.REFERENCES.

  1. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes, estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047-53.
  1. Mangala R, Mhaskar R, Mhaskar A, Lillian T, Narayanan R. Perinatal outcome in pregnancy complicated by Diabetes mellitus. Intnl J Diab Dev Countries 1991;11:22-4.
  1. Diabetes and Pregnancy Group, France. French multicentric survey of outcome of pregnancy in women with pregestational diabetes. Diabetes Care 2003;26:2990-3.
  1. American Diabetes Association. Preconception care of women with diabetes. Diabetes Care 2004;27(Suppl 1):S76-8.
  1. ShefaliAK, Kavitha M, Deepa R, Mohan V. Pregnancy outcomes in pre-gestational and gestational diabetic women in comparison to non-diabetic women-A prospective study in Asian Indian mothers (CURES-35). JAPI 2006;54:613-18.
  1. The Diabetes Control and Complications Trial Research Group. Pregnancy outcomes in the Diabetes Control and Complications Trial. Am J Obstet Gynecol 1996;174:1343-53.
  1. Banerjee S, Ghosh US, Banerjee D. Effect of tight glycaemic control on fetal complications in diabetic pregnancies. JAPI 2004;52:109-13.
  1. Kerssen A, De Valk HW, Visser GHA. Increased second trimester maternal glucose levels are related to extremely large for gestational age infants in women with type 1 diabetes. Diabetes care 2007;30:1069-74.
  1. Herranz L, Pallardo LF, Hillman N, Vaquero PM, Villarroel A, Fernandez A. Maternal third trimester hyperglycemic excursions predict large-for-gestational-age infants in type 1 diabetic pregnancy. Diabetes Research and Clinical Practice 2007;75:42-6.
  1. Tinker A, Paul V, Ruben J. The right to a healthy newborn. Intl J of Gynecol and Obstet 94(3):269-76.
  1. Sack DB, Bruns De, GoldsteinDE, Maclaren NK, Mcdonald JM, Parott M. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clinical Chemistry 2002;48(3):436-72.
  1. Evers IM, de Valk HW, Visser GHA. Risk of complications of pregnancy in women with type 1 diabetes: nationwide prospective study in the Netherlands. BMJ, doi:10.1136/bmj.38043.583160.EE(published 5 April 2004).
  1. Worth R, Potter JM, Drury J, Fraser RB, Cullen DR. Glycosylated hemoglobin in normal pregnancy: a longitudinal study with two independent methods. Diabetologia 1985;28:76-9.
  1. Vintzileos AM, Thompson JP. Glycohemoglobin determinations in normal pregnancy and in insulin-dependent diabetics. Obstet Gynecol 1980;56:435-39.
  1. Nielsen LR, Ekbom P, Damm P, Glumer C, Frandsen MM, Jensen DM et al. HbA1c levels are significantly lower in early and late pregnancy. Diabetes Care 2004;27:1200-01.
  1. Radder JK, Roosmalen JV. HbA1c in healthy, pregnant women. The Netherlands Journal of Medicine 2005;63(7):256-59.
  1. Fadel HE, Hammind SD, Huff TA, Harp RJ. Glycosylated hemoglobins in normal pregnancy and gestational diabetes mellitus. Obstet Gynecol 1979;54:322-26.
  1. O’Shaughnessy R, Russ J, Zuspan FP. Glycosylated hemoglobins and diabetes mellitus in pregnancy. Am J Obstet Gynecol 1979;135:783-90.
  1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004;27(Suppl 1):S5-S10.
  1. Jovanovic L, Peterson CM. Optimal insulin delivery for the pregnant diabetic patient. Diabetes Care 1982;5(suppl 1):24-37.
  1. Mills JL, Jovanovic L, Knopp R, Aarons J, Conley M, Park E et al. Physiological reduction in fasting plasma glucose concentration in the first trimester of normal pregnancy: the diabetes in early pregnancy study. Metabolism 1998;48:1140-44.
  1. Balaji V, Balji SM, Srinivasan A, Suganthi S, Suresh S, Veeraswamy S. A1c in gestational diabetes mellitus in Asian Indian women. Diabetes Care 2007;30:1865-7.

9. Signature of the candidate :

10. Remarks of the guide : Infrastructure for the study is available. The student is capable

of carrying out the study. It is important to create an awareness of HbA1c as a

marker of glycemic control and to study the HbA1c pregnancy specific ranges.

11. Name and designation of

11.1 Guide : DR. SULTANA FURRUQH

PROFESSOR AND HEAD

DEPARTMENT OF BIOCHEMISTRY

ST. JOHN’S MEDICALCOLLEGE

BANGALORE.

11.2Signature :

11.3Co-guides : DR. ANNAMMA THOMAS

PROFESSOR AND HEAD

DEPARTMENT OF OBSTETRICS AND GYNECOLOGY

ST. JOHN’S HOSPITAL

BANGALORE.

Signature :

DR. VAGEESH AYYAR

ASSOCIATE PROFESSOR

DEPARTMENT OF ENDOCRINOLOGY

ST. JOHN’S HOSPITAL

BANGALORE.

Signature :

11.4 Head of the

department : DR. SULTANA FURRUQH

PROFESSOR AND HEAD

DEPARTMENT OF BIOCHEMISTRY

ST. JOHN’S MEDICALCOLLEGE

BANGALORE.

11.5 Signature :

12.1Remarks of the chairman and principal :

12.2 Signature:

PATIENT INFORMATION AND CONSENT FORM

STUDY OF USE OF MATERNAL GLYCATED HEMOGLOBIN IN THE SCREENING OF GESTATIONAL DIABETES.

Investigator : DR. CHRISHANTHI QUIRINE RODRIGUES

Institutional address : St. John’sMedicalCollege and Hospital, Sarjapur road,

Koramangala, Bangalore-560034.

Introduction :

You are requested to take part in a research study of Glycated hemoglobin in pregnancy.

Serum Glycated hemoglobin and serum glucose levels will be assessed in your blood sample as a part of the study.

Before agreeing to participate in this study, you will receive oral and written information about this study. Your participation in this study will be in the form of giving your blood samples during the study period.

Purpose of the study :

The purpose of this study is to assess the levels of Glycated hemoglobin in pregnancy and compare it to the non-pregnant state.

Study procedures :

If you choose to participate in the study, your blood sample will be collected in the second trimester for Glycated hemoglobin and serum glucose estimation.

Risks :

For most people, needle punctures for blood draws do not cause any serious problems. However, they may cause bleeding, bruising, discomfort, infections, and/or pain at the needle site or dizziness.

Participation in the study :

Your taking part in the study is entirely voluntary. You may refuse to take part in the study.