Hyperglycemia induces embryopathy, even in the absence of systemic maternal diabetes: an in vivo test of the fuel mediated teratogenesis hypothesis

Abstract

Embryonic exposure to excess circulating fuels is proposed to underlie diabetic embryopathy. To isolate the effects of hyperglycemia from the many systemic anomalies of diabetes, we infused 4 mg/min glucose into the left uterine artery of non-diabetic pregnant rats on gestation days (GD) 7-9. Right-sided embryos and dams exhibited no glucose elevation. Embryos were assessed on GD13, comparing the left versus right uterine horns. Hyperglycemic exposure increased rates of embryopathy, resorptions, and worsened embryopathy severity. By contrast, saline infusion did not affect any of these parameters. To assess for possible embryopathy susceptibility bias between uterine horns, separate dams were given retinoic acid (25mg/kg, a mildly embryopathic dose) systemically on GD7.5. The resultant embryopathy rates were equivalent between uterine horns. We conclude that hyperglycemia, even in the absence of systemic maternal diabetes, is sufficient to produce in vivo embryopathy during organogenesis.

Keywords: Animal model; Diabetes; Embryopathy; Fuel-mediated teratogenesis; Hyperglycemia; Teratogenesis.

Figure 1. Experimental design and maternal characteristics by experimental group

(A) Timeline: A catheter infusing into the left uterine artery was placed on gestational days 1–2. (B,C) There were three experimental groups of dams: glucose (GLU, black line, filled circle), saline (SAL, grey line, open circle), and retinoic acid (RA, dotted line, open square). GLU and SAL dams received glucose (4 mg/min) or normal saline, respectively, infused via the catheter on gestational days 7–9. Retinoic acid (25 mg/kg) was administered to RA dams intraperitoneally on gestational day 7.5. A portion of RA dams also received a catheter with or without saline infusion. Embryos were assessed on gestational day 13 (GLU and RA) or 14 (SAL). (B) Maternal weight change from baseline (gestational day 1) by experimental group. (C) Maternal systemic blood glucose by experimental group. The day 9 blood glucose was measured prior to discontinuing infusions. *p<0.05 for difference between RA and SAL mothers by ANOVA and Tukey's HSD post-doc analysis. Note that at day 1 the three groups had not yet received differing treatments. There were no other statistical differences between groups.

Figure 2. Comparison of embryopathy rates between uterine horns

(A) Mean embryopathy rates in the left (black bars) and right (grey) uterine horns. *P<0.001 (B) Null distributions for the difference between right and left uterine horn embryopathy rates, determined by random permutation testing, are shown in grey for the three experimental groups. The actual observed embryopathy rate difference is shown as a dark arrow.

Figure 3. Comparison of embryopathy rates by differing exposures

Embryopathy rates by exposure: none (Ø), saline (SAL), hyperglycemia (↑GLU), or retinoic acid (RA). * P<0.05, # P<0.01 for the indicated effect by 1-way ANOVA and Tukey's HSD post-doc analysis.

Figure 4. Example micrographs across a range of embryopathy severity outcomes at gestational day 13

(A) Normal embryo. (B) Minor embryonic malformation, specifically disrupted caudal formation. (C) Major embryonic malformation, specifically a hindbrain neuropore defect (arrows). (D) Multiple apparent embryonic malformations. (E) Resorption, specifically empty amnion.

Figure 5. Embryopathy severity

(A) Comparison of mean embryopathy severity scores between left (black) and right (grey) uterine horns. *p<0.001 for difference between horns by random permutation testing. (B) Comparison of mean embryopathy severity scores between embryos grouped by exposure. * p<0.05 for indicated effect by ANOVA. (C) Distribution of embryopathy classes among embryos from left and right uterine horns. Embryos were classified as being normal (grey), or having a minor malformation (small cross-hatch), major malformation (white), multiple malformations (large cross hatch), or undergoing resorption (black). # p<0.005 for difference between left and right uterine horns by random permutation testing on either resorptions.