Abstract: Introduction:

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1 The Influence Of Maternal Diabetic Control And Insulin Antibodies On Fetal Pancreatic β- Cell Function And Neonatal Complications In Infants Of Insulin Treated Diabetic Mothers. Abdel Halim Badr El Din, Iman Marzouk, Hesham Ghazal, Salah Marzouk*, Karim Aref, and Mona Saleh. From the departments of Pediatrics, and Clinical pathology, Faculty of Medicine, Alexandria University, Egypt. Abstract: The current work aimed at studying the influence of maternal diabetic control on fetal β cell function and neonatal morbidity. It was conducted on 30 insulin treated diabetic mothers and their neonates who were matched with 15 infants of non diabetic mothers (INDMs) for gestational age, mode of delivery, and Apgar score. They were subjected to clinical assessment and laboratory investigations including estimation of cord blood insulin Abs and C- peptide and determination of serum levels of glucose, calcium, magnesium, hematocrit, and bilirubin. It was found that diabetic mothers had higher blood HbA1c concentrations than non diabetic mothers. Also, maternal HbA1c concentrations were proportionally correlated to the cord blood insulin Abs, and C- peptide levels; also to the birth weights of. There was an association between increased cord blood insulin Abs among and the development of macrosomia and hypoglycemia. Also, there was an increase in β cell function with the increase of cord blood insulin Ab level. Strict antenatal control of diabetes is strongly recommended to decrease the frequency of macrosomia and neonatal hypoglycemia. The use of purified insulin preparations with minimal immunogenicity would be the best. Introduction: Diabetes Mellitus and pregnancy have interrelated effects that can cause both, physiological and pathological disturbances in the mother and her offspring. Throughout pregnancy, placenta, either passively or actively permits, facilitates and adjusts the transfer of a wide range of substances from the mother to the fetus. Because glucose is transported across the placenta by facilitated diffusion, the glucose concentration gradient between maternal and fetal arterial blood is a major determinant of its transport which is also regulated by maternal insulin concentration and placental insulin receptors. (1) The fetal pancreas responds to glucose load and/ or maternal insulin antibodies by developing beta cell hypertrophy and hyperinsulinemia. Insulin is produced in pancreatic β - cell by cleavage of its single- chain polypeptide precursor - proinsulin- into insulin, the connecting C peptide and two pairs of basic amino acids. Insulin and C peptide are subsequently secreted into portal circulation in equimolar quantities along with a small amount of proinsulin and the intermediate form. Now, C peptide measurements create a new approach for investigating pancreatic β cell function. (2,3) Under the influence of increased circulatory insulin concentration and glucose flooding, the fetus copes by converting glucose to fat for storage in adiposites and increasing hepatic storage of carbohydrate and other nutrients. It has been suggested that the transplacental passage of insulin antibodies may play a role in the occurrence of fetal morbidity, however such effects of insulin antibodies are still controversial.(4) The aim of this work was to assess the relation between maternal glycosylated hemoglobin concentrations, fetal insulin antibodies, and C peptide levels and the occurrence of some neonatal morbidities among infants of insulin treated diabetic mothers (). Alexandria Journal of Pediatrics; Volume 12, Number 1; January

2 Subjects and Methods: The study included 30 insulin treated diabetic mothers and their neonates who were matched with 15 infants of non diabetic mothers (INDMs) for gestational age, mode of delivery, and one- and five- minute Apgar score. All neonates were clinically examined and their weight and length measurement. Infants with birth weights 4.2 kgm were considered macrosomic. Birth weight index (BWI) was calculated as actual B.wt / B.wt at 50 th percentile for the corresponding age and sex. (5) Laboratory procedures: Cord blood C peptide and insulin antibodies levels by radioimunoassay.(6,7) Blood glucose levels 1-2 hours after birth. Hypoglycemia was diagnosed when blood glucose< 35 mg/dl in full term babies, and < 25mg/dl in preterm babies were present. (8) Results: Statistical analysis of the data proved that: 1. Diabetic mothers had higher blood HbA1c concentrations than non diabetics. Three fourth of the mothers had poor diabetic control during the last two months of pregnancy as the HbA1c levels of 22 of them exceeded the 95% confidence limit ( 8.47% ) (table I ). 2. Infants of diabetic mothers had significantly higher birth weights and birth weight indices than INDMs.Birth weights and B.wt indices were significantly higher among with uncontrolled than those with controlled diabetic state. Also, there were positive correlations between maternal HbA 1c and both, actual birth weights, and birth weight indices of their neonates ( table II, figures 1,2 ). 3. Not only had higher blood insulin antibodies and C- peptide levels than INDMs, but also with poor maternal diabetic state had higher levels than with fair maternal diabetic state. Although there was no significant difference between cord blood insulin antibodies among with controlled diabetic state and INDMs, the difference was significant in respect to their C peptide levels. There were significant correlations between maternal HbA1c concentrations and both, cord blood insulin Abs and C Venous hematocrit (Ht) 2-4 hours after birth. Neonatal polycythemia was defined as Ht 65%. (9) Serum Ca and Mg levels at 24 hours after birth. Hypocalcemia & hypomagnesemia were diagnosed when serum levels 7 mg/dl and < 1.2 mg /dl respectively were included.(10) Serum bilirubin during the first three days of life. Hyperbilirubinemia was diagnosed when serum bilirubin levels>12 mg/dl in full term, and > 14 mg/dl in pre term babies were present. (8) Maternal glycosylated hemoglobin HbA 1c concentrations were estimated by microcolumn assay. The 95% confidence limit was calculated as mean+2sd of non diabetic HbA 1c values. (11) HbA1c concentrations 95% confidence limit was considered elevated due to poor diabetic control in the last two months of pregnancy.(12) peptide levels. Also, cord blood insulin Abs and C peptide levels were proportionally correlated (table III). 4. Frequent laboratory investigations -during the first 72 hours after birth-among infants of diabetic and non diabetic mothers revealed : either with controlled or uncontrolled diabetic state had lower serum calcium and magnesium, and higher venous hematocrit levels than INDMs. Only with uncontrolled diabetic state had lower blood glucose and higher serum bilirubin levels than INDMs (table IV). either with controlled or uncontrolled diabetic state had significantly more frequent macrosomia, hypoglycemia, hypocalcemia, hypomagnesemia, polycythemia, and hyperbilirubinemia than INDMs (table V). Both, hypoglycemic and macrosomic had significantly higher levels of cord blood insulin antibodies than normoglycemic and non macrosomic respectively (tables VI,VII). There were significant inverse correlation between blood glucose levels among and their cord blood C peptide levels, actual birth weights, and birth weight indices. (figures 3,4,5) Table I: HbA 1c concentrations among diabetic and non diabetic mothers : Diabetic mothers Non diabetic mothers F test values Alex. J. Pediatr. 12(1), Jan

3 HbA1c % 8.47 <8.47 n=22 n=8 n=15 Range Mean ± SD 11.7± ± ±1.09 * = Significant at 5% level * (1,2) (1,3) Table II : Birth weights (kgm) & birth weight indices of the studied infants in respect to maternal diabetic control. INDMs F test values HbA1c 8.47 HbA1c<8.47 n=22 n=8 n=15 Birth weight range * Mean ± SD 4.6± ± ±0.40 (1,2)(1,3) (2,3) BWI range * (1,2) Mean ± SD 1.44± ± ±0.14 (1,3) (2,3) *Significant at 5% level. Table III: Cord blood insulin antibodies and C peptide levels among infants of diabetic and non diabetic mothers. INDMs F test values HbA1c 8.47 HbA1c<8.47 n=22 n=8 n=15 Insulin Abs range * Mean±SD 40.97± ± ±2.11 (1,2) (1,3) C peptide range * (1,2) Mean±SD 3.25± ± ±0.51 (1,3) (2,3) *Significant at 5% level Table IV:Laboratory data among infants of diabetic and infants of non diabetic mothers during the 1 st 72 hours of life. INDMs F test values HbA1c 8.47 HbA1c<8.47 n=22 n=8 n=15 Mean ± SD Mean ± SD Mean ± SD Blood glucose (mg/dl) 57.55± ± ± * (1,3) Serum Calcium (mg/dl) 7.15± ± ± * (1,3) (2,3) Serum 1.77± ± ± * (1,3) (2,3) Magnesium(mg/dl) Venous hematocrit (%) 69.77± ± ± * (1,3) (2,3) Serum bilirubin (mg/dl) 10.14± ± ± * (1,3) *Significant at 5% level. Table V: The frequency of some neonatal morbidities among the studied infants. HbA1c 8.47 n=22 HbA1c<8.47 n=8 INDMs n=15 N o % N o % N o % Significance of Z test * Macrosomia (1,3) (2,3) Hypoglycemia (1,3) (2,3) Hypocalcemia (1,3) (2,3) Hypomagnesemia (1,3) (2,3) Polycythemia (1,3) (2,3) Hyperbilirubinemia (1,3) (2,3) *Significant at 5% level. Table VI: Cord blood C-peptide and insulin antibodies among the studied in respect to macrosomia Alex. J. Pediatr. 12(1), Jan

4 c-peptide (ng/ml): range Mean ± S.D Insulinantibodies(%):range Mean ± SD *significant at 5% level Macrosomic n= ± ± 27.3 non macrosomic n= ± ± 29.8 t test value * Table VII: Cord blood C-peptide and insulin antibodies among the studied in respect to hypoglycemia Hypoglycemic Normoglycemic t test value (n=11 ) (n=19 ) c-peptide: range (mg/dl) Mean±S.D Insulin Abs(%) range * Mean±S.D * significant at 5% level Figure 1. The proportional correlation between maternal HbA1c & birth weights of Figure 2. The proportional correlation between maternal HbA1c & birth weight indices of Alex. J. Pediatr. 12(1), Jan

5 Figure 3. The inverse correlation between cord blood C-peptide levels among And their blood glucose levels 1-2 hours after birth Figure 4. The inverse correlation between birth weights of and their Blood glucose levels, 1-2 hours after birth Figure 5. The inverse correlation between birth weights indices of and their Blood glucose levels, 1-2 hours after birth Alex. J. Pediatr. 12(1), Jan

6 Discussion: The association of diabetes mellitus with pregnancy still carries a great risk to the fetus and newborn, in spite of marked reduction in rates of neonatal morbidity and mortality, which are mainly due to fetal hyperinsulinemia. ( 1 ) As all the diabetic mothers included in this study were class B insulin treated, both infants of diabetic and non diabetic mothers were matched for gestational age, mode of delivery, and Apgar score, hence the presence of any difference between them will be attributed mainly to the state of maternal diabetic control. The significantly elevated HbA1c levels among diabetic than non diabetic mothers were in agreement with the previous works. Also the poor diabetic control in three fourth of our diabetic mothers-evidenced by HbA1c concentration 95% confidence limit- could be explained by poor antenatal care and defective health education. In accordance to the previous works, the data of birth weights of the studied infants and their relation to the maternal diabetic control confirm the importance of estimating third trimester maternal HbA1c levels in predicting actual and relative birth weights in diabetic pregnancies. (1 2,13 ) A significantly higher mean cord blood insulin concentration among had been reported by Badr El Din et al ( 14 ) who did not prove the correlation between cord blood insulin and maternal HbA1c concentrations. They claimed transmitted anti-insulin antibodies from mothers to fetuses across the placenta to be a cause of impaired sensitivity of the immunoassay of insulin. In accordance to many previous works ( 3,15 ), not only had significantly higher cord blood C- peptide than INDMs but also those with poor diabetic control had significantly higher levels than with fair diabetic control. These findings were confirmed by the presence of proportional correlation between cord blood c peptide levels in our and HbA1c levels in their mothers. Such correlation signifies the importance of maternal diabetic control in lowering C- peptide levels among, hence decreasing their vulnerability to postnatal hypoglycemia and macrosomia. Petreson et al ( 16 ) found that who were treated with human insulin had lower C- peptide, less macrosomic and less prone to develop hypoglycemia than treated with other types of insulin. This partially explains the failure of C- peptide to reach a significant lower levels in non macrosomic and non hypoglycemic than macrosomic and hypoglycemic in the current work as none of our diabetic mothers were receiving human insulin due to financial reasons. However, the demonstrated association between elevated cord blood C- peptide and both decreased postnatal blood glucose levels and the increased actual and relative birth weights were in agreement with many studies.(15,16 ) In accordance to Edwin et al (18) INDMs included in this work showed low levels of cord blood insulin antibodies despite the fact that their mothers never received insulin. This was explained by the capacity of normal circulating CD5 lymphocytes to produce multivalent antibodies which bind multiple self antigens when stimulated by viral infection. These antibodies are of clinical significance only when their level is high and associated with high affinity to bind insulin. Being of the IgG class, insulin Abs were clearly demonstrated to cross the placenta appearing in fetal cord blood of all the studied, who not only had significantly higher insulin Abs than INDMs but also, infants of uncontrolled diabetic mothers had significantly higher insulin Abs than those of diabetic mothers with fair diabetic state. Also, the difference between the later and INDMs was not significant. Moreover, the demonstrated positive correlation between maternal HbA1c concentration and fetal cord blood insulin Abs signifies the importance of maternal diabetic control in lowering cord blood insulin Abs. Such correlation was expected as these Abs were found to adversely affect the metabolic control among diabetic patients. They can cause insulin resistance necessitating larger doses of exogenous insulin to achieve euglycemia. In addition, insulin-insulin Ab complexes release free insulin at inappropriate time resulting in widespread fluctuation in blood glucose level.(4,19) Several workers succeeded to link insulin Abs with the occurrence of macrosomia and hypoglycemia in. (3,17) We suggested that insulin Abs play a role in the occurrence of such morbidity as who developed macrosomia and /or hypoglycemia had significantly higher cord blood insulin Ab levels than those who did not develop such morbidity.in accordance to previous works, (8,9,10,20,21) our had lower Calcium and Magnesium, higher Alex. J. Pediatr. 12(1), Jan

7 hematocrit levels, and more frequent hypocalcemia, hypomagnesemia, and polycythemia than INDMs; but the development of these morbidities was not significantly correlated to the state of maternal diabetic control. The higher fequency of hyperbilirubinemia observed among may be due to ineffictive erythropoiesis or increased hemolysis or shorter life span of their red cells.(2) It is noteworthy that the proportional correlation between cord insulin Abs and C- peptide levels observed in were reported also by Heding et al (3) who suggested that such correlation is a consequence of actual increase in β-cell function in the presence of increased insulin Abs as C- peptide is not bound to those Abs. The mechanism by which insulin Abs affect neonatal morbidity is either directly or indirectly via complicating maternal diabetic control. Firstly, being of the IgG class, these Abs cross the placenta and neutralize endogenous insulin produced by the fetus, thus stressing the fetal pancreas to a degree greater than that attributed to hyperglycemia alone. Also, insulin Abs increase free plasma insulin in fetal blood as insulin-insulin Ab complexes cross the placenta and free insulin is gradually released from these complexes into the fetal circulation. Lastly, these Abs delay the clearance of insulin and prolong its biologic half life. All these factors further contribute to the fetal hyperinsulinemic state which is the main etiologic factor of macrosomia and hypoglycemia in. (4,19) To improve the outcome of diabetic pregnancy, and to reduce the frequency of neonatal macrosomia, and hypoglycemia there shoud be strict antenatal diabetic control of the mothers,and the use of purified insulin preparations with minimal immunogenecity. References: 1. London MB, Gabbe SG. Diabetes and pregnancy. Med Clin North Am 1998; 72: Morris FH. The infants of diabetic mothers. Fetal and neonatal pathophysiology. Pers Ped Path 1984; 8: Heding LG, Persson B,Stangenberg MB. β cell function in newborn infants of diabetic mothers.diabetologia 1980;19: Mylvaganam R, Stowers JM, Steel JM, Wallace J, Machendry JC, Wright AD. Insulin immunogenecity in pregnancy: Maternal and fetal studies. Diabetologia 1983;24: Berk AM, Mimouni F, Miodovnik M, Hertzberg V, Valuck J. Macrosomia in infants of insulin-dependent diabetic mothers. Pediatrics 1989;83: Block MR, Pilds RS, Mossabhoy NA, Steiner DF, Rubenstein AH. C- peptide immunoreactivity (CPR) :A new method for studying infants of insulin -treated diabetic mothers. Pediatrics 1974;53: Christiansen AH. Radioimunoelectropheresis in the determination of insulin binding to IgG; Methodological studies. Hor Metab Res 1973;5: Perelman CH. The infant of diabetic motherpathophysiology and management. Primary Care 1983;10: Mimouni F, Midovnik M, Siddiqui A, Bulter JB, Harolde J, Tsang RC. Neonatal polycythemia in infants of insulin dependent diabetic mothers. Obstet Gynecol 1986;68: Mimouni F, Tsang RC, Hertzberg VS, Medovnik M. polycythemia, hypomagnesemia, and hypocalcemia in infants of diabetic mothers. Am J Dis Child 1986;140: Kaplan LA, Cline D, Gartside P. Hemoglobin A1c in hemolysate from healthy and insulin dependent diabetic childrin as determint with a temperature controllrd mini- column assay. Clin Chem 1982;28: Javonovic L, Petresson C, SaxonBB, Dawood MY, Saudek CD. Feasibility of maintaining normal glucose profile in insulin dependent pregnant diabetic women. Am J Med 1980;68: Ylinen K, Raivio K, Temro K. Hemoglobin A1c predicts the perinatal outcome in insulin dependent diabetic pregnancies. Br J Obstet Gynecol 1981;88: Badr El-Din AH, Abdel Aal A, Abdel Latif MJ, Marzouk IM. The relationship between maternal glycosylated hemoglobin and birth weight cord blood insulin and cortisol level in diabetic pregnancy. Alex J Pediatr 1992; 6: Sosenko IR, Kitzmiller JL, Loo SW, Blix P, Rubenstein A, Gabby KH. Infant of diabetic mothers: Correlation of increased cord C peptide levels with macrosomia and hypoglycemia. New Engl L Pediatr 1980; 301: Peterson LJ, Kitzmiller JL, Choles F. Randomized trial of human versus animal insulin in diabetic pregnant women: Improved glycemic control, not fewer antibodies to insulin, influencees birth weight. Am J Obstet Gynecol 1992; 13: Felluca F, Moldonato A, Lavicoli M, Di Rollo G, Di Biase N, Napoli A, De Vecchis P, Scuillo E, Gerlini G, Pachi A. Influence of maternal metabolic control and insulin antibodies on neonatal complications in infants Alex. J. Pediatr. 12(1), Jan

8 of diabetic mothers. Diabetic Res Clin Practice 1989; 7: Edwin F, Biegel AA, Lurr KL, Huffered S. Presence of insulin auto-antibodies as regular feature of non diabetic repertoire of immunity. Diabetes 1991; 40: Di Mario V, Felluca F, Gargiulo P. Insulin -anti-insulin complexes in diabetic women and their neonates. Diabetologia 1980: 19: Marzouk IM, Badr El Din AH, Marzouk SA, Basha Y. The relatin between the state of diabetic control in mothers and calcium changes in their offspring. Alex J Pediatr 1995;3: Marzouk IM, El Domiaty BA, Marzouk SA, Seliem EH. Cord blood erythropoietin level and its relation to the maternal diabetic control. Alex J Pediatr 1995; 3: Alex. J. Pediatr. 12(1), Jan