Maternal diabetes mellitus (DM) affects early organogenesis. Metabolic disorders of DM are associated with a depleted zinc status. This study evaluated the effect of maternal DM on cardiac development of rat fetuses and protective roles of prenatal zinc versus insulin supplementation. Pregnant rats were divided into 4 groups ((I) control, (II) STZ-induced DM, (III) STZ-induced DM treated with Zn, and (IV) STZ induced DM treated with insulin), all sacrificed on GD 20. Fetal heart weight of diabetic rats showed significant decrease compared to controls (
Global prevalence of DM in 2010 among adults has been reported to be 6.4% [
Several studies considered hyperglycemia as an independent risk factor directly causing cardiac damage, leading to diabetic cardiomyopathy [
Alterations of antioxidant micronutrient status have been reported in subjects with type 1 or 2 diabetes mellitus [
Therefore, this study was conducted to assess the effect of maternal diabetes on the cardiac development of albino rat fetuses and the roles of prenatal zinc/or insulin administration in counteracting this effect.
Experimental study included four groups (6/group) of female albino rats. Group (I) was the control group and groups (II), (III), and (IV) were the diabetic groups. Thirty-eight fetuses were collected from the pregnant mothers of each group on Gestational Day (GD) 20.
Diabetes was induced in the virgin female albino rats of groups (II) to (IV) by a single dose of intraperitoneal administration of streptozotocin (40 mg/kg). Blood glucose was measured 4 days after STZ injection from the tail vein using ultra one touch device and rats with blood glucose levels exceeding 200 mg/dL were considered diabetic.
After induction of diabetes, mating was carried out by placing two female rats with one male rat in a cage overnight. Pregnancy was verified by the presence of spermatozoa in the vaginal smears, taken from the mated females, which denotes the first day of gestation (GD1) and the assignment of groups were as follows.
In all cases, the pregnant rats were sacrificed by an overdose of ether on GD 20 and the fetuses were obtained through caesarian section.
The fetuses were dissected out; the viable fetuses and placentas were weighed. Fetal hearts were dissected out, weighed, collected, and fixed in 4% paraformaldehyde in the phosphate buffered saline for 1~6 hours. Then, the fixed hearts were embedded by paraffin and sectioned following routine practice. The sections were manipulated to be 5
H&E staining was performed using routine method. Sections were examined by light microscope to comment on the general architecture of the myocardial tissue.
Paraffin embedded sections were also analyzed by caspase-3 immunostain (present in apoptotic cells) and polyclonal anti-ki 67 antibody (present only in cycling cells to estimate the degree of cardiomyocyte proliferation).
No change: normal cardiac microscopy. Mild change: vacuolar degeneration in 5–15% of cells. Moderate change: vacuolar degeneration in 16–35% of cells. Marked change: vacuolar degeneration in more than 35% of cells.
The apoptotic index was determined as follows [ Grade I: apoptotic cells not detected or AI < 1%. Grade II: AI < 5%. Grade III: AI: 5–10%. Grade IV: AI > 10%.
The proliferating cells (KI67 immunohistochemically positive cells) were counted in five high-power fields (400x magnification) for each section; then, proliferation index was calculated by the percentage of positive cells in each field as follows [ Low proliferation KI67 index ≤ 15%. Intermediate proliferation KI67 index 16–30%. High proliferation KI67 index > 30%.
The data were analyzed using SPSS version 16.0. Quantitative parametric variables were presented by mean and standard deviation (SD) and compared by Student’s
A statistically significant increase in both maternal and fetal random blood glucose was observed in STZ-induced DM group when compared to the control, STZ-induced DM treated with zinc, and STZ-induced DM treated with insulin groups (Table
Mean ± SD of maternal and fetal random blood glucose levels in the different groups.
Parameter | Group | ANOVA | ||||
---|---|---|---|---|---|---|
C | STZ-DM | STZ-DM + Zn | STZ-DM + In |
|
|
|
Maternal random blood glucose (mg/dL) | 108.33 ± 11.25 | 360.00 ± 63.48 |
180.00 ± 38.34 |
115.83 ± 32.00 |
49.5 | <0.00001 |
|
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Fetal random blood glucose (mg/dL) | 96. 67 ± 7.53 | 334. 67 ± 63.19 |
123.17 ± 40.31 |
91.67 ± 17.52 |
54.2 | <0.00001 |
C: control group, STZ-DM: streptozotocin-induced diabetic group, STZ-DM + In: streptozotocin-induced diabetic treated with insulin group, and STZ-DM + Zn: streptozotocin-induced diabetic treated with Zn group.
Moreover, the maternal random blood glucose showed a statistically significant increase in STZ-induced DM treated with zinc group when compared to the control one and STZ-induced DM treated with insulin group (Table
Table
Pregnancy outcome and embryolethality in the different groups.
Parameter | Group | Chi-square |
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---|---|---|---|---|---|---|---|---|---|---|
C | STZ-DM | STZ-DM + Zn | STZ-DM + In | |||||||
No. | % | No. | % | No. | % | No. | % | |||
Total rats | 6 | 100 | 12 | 100 | 8 | 100 | 8 | 100 | — | — |
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Mortality rate | 0 | 0 | 6 |
50 | 2 |
25 | 2 | 25 |
66.67 | <0.00001 |
|
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Total number of corpora lutea (in metrial gland) | 39 | 50 |
42 | 42 | 2.99 | 0.56 | ||||
|
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Resorptions | 1 | 2.6 | 12 |
24 | 4 | 9.5 | 2 | 4.8 | 28.62 | <0.00001 |
Total number of fetuses | 38 | 97.4 | 38 | 76 | 38 | 90.5 | 40 | 95.2 | ||
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Live fetuses | 38 | 100 | 31 | 81.6 | 36 | 94.7 | 36 | 90 | 23.35 | 0.000034 |
Dead fetuses | 0 | 0 | 7 |
18.4 | 2 | 5.3 | 4 | 10 |
C: control group, STZ-DM: streptozotocin-induced diabetic group, STZ-DM + In: streptozotocin-induced diabetic treated with insulin group, and STZ-DM + Zn: streptozotocin-induced diabetic treated with Zn group.
The differences in these parameters (number of corpora lutea and rate of resorption) between STZ-induced DM group and STZ-induced DM treated with both zinc and insulin groups were proved to be insignificant (Table
Regarding number of dead fetuses, a statistically significant increase was found in STZ-induced DM group when compared to the control group, while a statistically nonsignificant difference was observed between STZ-induced DM group and both STZ-induced DM treated with zinc and STZ-induced DM treated with insulin groups (Table
Mean ± SD of the maternal, placental, fetal, and fetal heart weights in the different groups.
Parameter | Group | ANOVA | ||||
---|---|---|---|---|---|---|
C | STZ-DM | STZ-DM + Zn | STZ-DM + In |
|
|
|
Maternal weight gain (gm) | 47.5 ± 3.08 | 33.83 ± 2.93 |
41.33 ± 3.98 |
38.17 ± 2.79 |
19.04 | <0.00001 |
Placental weight (gm) | 0.565 ± 0.081 | 0.624 ± 0.211 | 0.545 ± 0.092 |
0.540 ± 0.087 |
3.39 | 0.020 |
Fetal weight (gm) | 3.29 ± 0.21 | 2.98 ± 0.33 |
3.16 ± 0.44 | 3.21 ± 0.42 |
4.89 | 0.003 |
Fetal heart weights (gm) | 0.479 ± 0.062 | 0.424 ± 0.071 |
0.461 ± 0.095 | 0.475 ± 0.075 |
4.34 | 0.006 |
C: control group, STZ-DM: streptozotocin-induced diabetic group, STZ-DM + In: streptozotocin-induced diabetic treated with insulin group, and STZ-DM + Zn: streptozotocin-induced diabetic treated with Zn group.
Regarding maternal weight gain, a statistically significant increase was proved in the control group compared to the STZ-induced DM, STZ-induced DM treated with zinc, and STZ-induced DM treated with insulin groups. On the contrary, a statistically significant decrease was found in the maternal weight gain in STZ-induced DM group when compared to STZ-induced DM treated with zinc and the STZ-induced DM treated with insulin groups (Table
A statistically nonsignificant difference was found on comparing fetal heart weight of the control group with both the STZ-induced DM treated with zinc and the STZ-induced DM treated with insulin groups (Table
Moreover, on comparing the fetal heart weight of the STZ-induced DM treated with zinc group with that of the STZ-induced DM treated with insulin group, a statistically nonsignificant difference was evident (Table
Photomicrograph of H&E (×400) sections in the fetal myocardium. (a) Control group showing normal striated pattern of the cardiomyocytes with centrally located nuclei (arrow) and eosinophilic cytoplasm (double arrow). (b) STZ-induced DM group showing myocardial disarray, marked vacuolar degeneration of the cardiomyocytes (V), fragmentation of the nuclei of cardiomyocytes (arrow) and pyknotic changes of the nuclei (arrow head), congested blood vessels (C), and discontinuation of the vessel wall (dashed arrow) with the presence of subendocardial thickening (double arrow). (c) STZ-induced DM treated with zinc group showing restored architecture of cardiomyocytes with the presence of mild vacuolar degeneration (V). (d) STZ-induced DM treated with insulin group showing focal improvement of the myocardium with the presence of vacuolar degeneration (V) and myocardial hemorrhage (H) with extravasated red blood cells within the myocardial tissue (arrow).
Myocardial sections of the control group stained with caspase-3 immunostain showed negative brown cytoplasmic immunoreaction (Figure
Photomicrograph of caspase-3 immunohistochemical staining (×400) sections in the fetal myocardium. (a) control group. (b) STZ-induced DM group. (c) STZ-induced DM treated with zinc group. (d) STZ-induced DM treated with insulin group. Shown is positive brownish cytoplasmic immunohistochemical staining with caspase-3 in group (b) which seems to be downregulated in both group (c) and group (d); this brownish cytoplasmic immunohistochemical staining is almost absent in group (a).
A photomicrograph of KI67 immunohistochemical staining (×400) sections in the fetal myocardium. (a) Control group showing dense positive brown nuclear immunohistochemical staining with KI67. (b) STZ-induced DM group showing negative nuclear immunohistochemical staining with KI67. (c) STZ-induced DM treated with zinc group showing moderate positive nuclear immunohistochemical staining with KI67. (d) STZ-induced DM treated with insulin group showing minimal positive nuclear immunohistochemical staining with KI67.
The myocardial sections stained with caspase-3 immunostain showed positive brown cytoplasmic caspase-3 immunoreactivity (Figure
Very few focal areas of hemorrhage and vascular congestion were also seen (Figure
Myocardial sections stained with caspase-3 immunostain showed minimal positive brown cytoplasmic caspase-3 immunoreactivity (Figure
Myocardial sections stained with caspase-3 immunostain showed moderate positive brown cytoplasmic caspase-3 immunoreactivity (Figure
Percentage of frequency distribution of vacuolar degenerative changes in the different groups.
Parameter | Group | |||
---|---|---|---|---|
C | STZ-DM | STZ-DM + Zn | STZ-DM + In | |
No change (%) | 95 | 0 | 2 | 2 |
Mild change (%) | 5 | 5 | 80 | 25 |
Moderate change (%) | 0 | 15 | 13 | 55 |
Marked change (%) | 0 | 80 | 5 | 18 |
Total (%) | 100 | 100 |
100 |
100 |
Chi-square 637.9.
C: control group, STZ-DM: streptozotocin-induced diabetic group, STZ-DM + In: streptozotocin-induced diabetic treated with insulin group, and STZ-DM + Zn: streptozotocin-induced diabetic treated with Zn group.
Percentage frequency distribution of apoptotic index (AI) in caspase-3 immunohistochemical stained myocardial sections in the different groups.
Parameter | Group | |||
---|---|---|---|---|
C | STZ-DM | STZ-DM + Zn | STZ-DM + In | |
Grade I AI < 1% | 88 | 0 | 8 | 5 |
Grade II AI < 5% | 10 | 2 | 72 | 25 |
Grade III AI: 5–10% | 2 | 23 | 15 | 55 |
Grade IV AI > 10% | 0 | 75 | 5 | 15 |
Total (%) | 100 | 100 |
100 |
100 |
Chi-square 533.86.
C: control group, STZ-DM: streptozotocin-induced diabetic group, STZ-DM + In: streptozotocin-induced diabetic treated with insulin group, and STZ-DM + Zn: streptozotocin-induced diabetic treated with Zn group.
Percentage frequency distribution of proliferation index in KI67 immunohistochemical stained myocardial sections in the different groups.
Parameter | Group | |||
---|---|---|---|---|
C | STZ-DM | STZ-DM + Zn | STZ-DM + In | |
Low proliferation KI67 index ≤ 15% | 12 | 68 | 23 | 27 |
Intermediate proliferation KI67 index 16–30% | 25 | 17 | 47 | 46 |
High proliferation KI67 index >30% | 63 | 15 | 30 | 27 |
Total (%) | 100 | 100 |
100 |
100 |
Chi-square 113.17.
C: control group, STZ-DM: streptozotocin-induced diabetic group, STZ-DM + In: streptozotocin-induced diabetic treated with insulin group, and STZ-DM + Zn: streptozotocin-induced diabetic treated with Zn group.
In the current study, the maternal blood glucose levels of diabetic (360.00 mg/dL) and Zn-supplemented diabetic (180.00 mg/dL) groups were significantly higher than those of the control (108.33 mg/dL) and insulin treated (115.83 mg/dL) groups. Moreover, the fetal blood glucose level of diabetic (334.67 mg/dL) group was significantly higher than that of the control (96.67 mg/dL), Zn-supplemented diabetic (123.17 mg/dL), and insulin treated (91.67 mg/dL) groups. We observed that administration of zinc during pregnancy improved both the maternal and fetal random blood sugar but did not reach the levels of the control and insulin treated groups. In parallel to our study, the study of Kumar et al. in Singapore reported that maternal blood glucose levels of diabetic and zinc-supplemented diabetic groups were higher than those of the control group [
The present study revealed that STZ-induced DM group showed a statistically significant increase in the mortality rate compared to the control, STZ-induced DM treated with zinc, and STZ-induced DM treated with insulin groups. Additionally, resorption rate was found in significantly higher level in the STZ-induced DM group compared to the control group. Administration of both zinc and insulin resulted in improvement of the mortality rate and the rate of resorption. However, this improvement did not reach that of the control group.
Regarding the number of dead fetuses, a significantly higher level was noted in STZ-induced DM group compared with the control group, with statistically nonsignificant difference observed compared with STZ-induced DM treated with zinc group and STZ-induced DM treated with insulin group. These data were supported by several studies [
The reduction in number of embryos observed in diabetic pregnancies could be due to either reduced fecundity or early embryonic lethality resulting from a higher malformation rate in the embryos of diabetic mothers compared with those of nondiabetic mothers as explained by Phelan et al. [
Zinc is a transition metal which may protect against oxidation of vital compounds and inhibit production of reactive oxygen species [
Additionally, embryonic lethality rate was found to be decreased by the administration of insulin during pregnancy which corrects the hyperglycemia, a result that seems concordant with the study of [
In the current study, there was no weight difference between any of the experimental groups on the day of conception. In general, all groups gained weight during pregnancy. The control group, STZ-induced DM treated with zinc group, and STZ-induced DM treated with insulin group showed greater increase in maternal body weight than the STZ-induced DM group. The increase in body weight in STZ-induced DM treated with zinc group was less than the control group; these results coincide with the studies of Kumar et al. which observed that all studied groups gained weight during pregnancy and the STZ-induced DM treated with zinc group was higher than the diabetic group [
The present study also showed that fetal body weight had significantly lower value in the fetuses of diabetic mothers compared with fetuses of control, STZ-induced DM treated with zinc, and STZ-induced DM treated with insulin groups. This agrees with the studies of Simán et al. [
Some of the reduced fetal weight observed in the present study possibly occurs due to the production of reactive oxidative species, which was preventable with antioxidant treatment, whereas the remaining growth retardation may have resulted from other non-ROS-related mechanisms as the availability of nutrients and oxygen to the fetus, intrauterine insults, and a variety of growth factor and proteins of maternal, fetal, and placental origin most of which are disturbed with diabetic pregnancy as explained by Kumar et al. [
By comparing the fetal body weight in the zinc and insulin treated groups with the control group, we found insignificant difference indicating the protective role of zinc and insulin in the diabetic embryopathy which coincides with the results of He et al. [
Regarding the placental weight, it showed greater values in the diabetic group when compared with the control, zinc treated, and insulin treated groups. This result is in match with the results of several studies as Eriksson et al. [
In addition, the current study revealed that the fetal and placental weights of the insulin treated diabetic group were nearly similar to those of the control group and this was in concordance with the study of Ericsson et al. [
On examination of the Hematoxylin and Eosin stained sections of the diabetic group, it revealed myocardial disarray accompanied with loss of the normal striated architecture of the cardiomyocytes with presence of characteristic degenerative changes of the nuclei in the form of pyknosis, karyorrhexis, and karyolysis. These results coincide with the results of Schneider et al. [
Moreover, most of the fetal myocardium of the diabetic group showed marked vacuolar degeneration of the cardiomyocytes, accompanied with marked vascular congestion, hemorrhage, subendocardial thickening, and vascular wall thickening. These results coincide with that of the Mexican study done by Manjarrez et al. which reported the presence of dilated sinusoids with endothelial lining in the myocardium, after application of STZ [
The insulin treated diabetic group showed only partial improvement but there still exist moderate vacuolar degenerative changes of the myocytes, congestion, and hemorrhage. Regarding zinc treated diabetic group, the fetal myocardium showed marked improvement in the myocytes, minimal degenerative changes, and restored architecture of normal myofibrils. The previous findings seem to be concordant with the study of Kumar et al. who found that the structure of the myocardium was better preserved in the hearts of diabetic rats treated with zinc. They reported reduced numbers and spread of cardiomyocytes degeneration, which mostly showed normal nuclei and homogeneous cytoplasm. The frequency of fibrotic scars and accumulation of interstitial and perivascular connective tissue were clearly diminished in the zinc-protected group. Moreover, there were restored normal myofibril architecture and orientation of myocytes with decreased cardiac anomalies [
The precise mechanism by which zinc antagonizes the cardiac teratogenic effects of diabetes remains uncertain. However, it is likely to be the result of an effect on several metabolic functions that are associated with antioxidation. Zinc protects cells from abnormal synthesis of nucleic acids and proteins, chromosomal defects, and impairment of cellular growth and morphogenesis [
Immunohistochemical staining with caspase-3 immunostain, which is an important indicator of apoptotic cell death, showed positive brown cytoplasmic caspase-3 immunoreactivity in the fetal heart of diabetic mothers as compared with control group (negative brown cytoplasmic immunoreaction). Minimal positive brown cytoplasmic caspase-3 immunoreactivity and moderate positive brown cytoplasmic caspase-3 immunoreactivity were observed in the zinc treated diabetic group and insulin treated diabetic groups, respectively. These results suggest that apoptosis via caspase-3 activation, which occurred in the myocardium of diabetic fetuses, can be minimized by administration of therapeutic doses of zinc and insulin during pregnancy. This is supported by the results of the studies done by Kumar et al. [
Apoptosis of cardiac muscle cells and endothelial cells has been observed in the heart of patients with diabetes [
On the other hand, immunohistochemical staining of the fetal heart sections with KI67 was done to assess the cellular proliferation. The current results revealed negative brown nuclear immunoreaction indicating minimal proliferative cells in the fetuses of the diabetic mothers compared to dense positive brown nuclear immunoreaction, moderate positive brown nuclear immunoreaction, and minimal positive brown nuclear immunoreaction in the control, zinc treated diabetic, and insulin treated diabetic groups, respectively. These results seem concordant with the results of Song et al. [
The present study revealed that embryonic cardiac tissues respond differently to the adverse environment created by maternal diabetes during pregnancy. We found that treating hyperglycemia by insulin ameliorates myocardial morphological abnormalities and partially inhibits myocardial cell death. Our study also suggests that Zn has antiapoptotic activity against glucose-induced myocardial disarrangement caused by decreased cellular proliferation and increased apoptotic level. The adverse effects of maternal diabetes on an unborn fetus could possibly be treated by insulin and Zn supplementation in the early stages of gestational diabetic pregnancy.
Gestational diabetes was associated with increased risk of fetal myocardial damage that might be caused by decreased cellular proliferation and increased apoptotic level. Treating diabetic pregnant females with both therapeutic doses of zinc and insulin was associated with improvement in myocardial integrity. However, STZ-induced DM treated with zinc group showed better improvement than STZ-induced DM treated with insulin group.
The authors declare that there is no conflict of interests regarding the publication of this paper.