In line with our previous findings35, fasting blood glucose levels were higher in non-pregnant Alx3-deficient than in wild type females, but they remained with normal range (<120 mg/dl) and never reached levels characteristic of overt diabetes, defined as fasting blood glucose concentrations higher than 250 mg/dl (Fig. 1A). Since non-pregnant Alx3-deficient females had only a mild hyperglycaemia, we sought to determine whether pregnancy in these animals was associated with increased basal blood glucose levels or severe glucose intolerance. Glucose tolerance tests demonstrated the presence of relatively mild glucose intolerance in Alx3-deficient non-pregnant females as compared with wild type non-pregnant controls (Fig. 1B). When tests were carried out in pregnant females, we found that wild type animals had developed a degree of glucose intolerance similar to that found in Alx3-deficient females (Fig. 1B), consistent with the normal metabolic adaptations of maternal glucose homeostasis during pregnancy37–39. In contrast, pregnancy did not aggravate further the glucose intolerance observed in Alx3-deficient females, which remained similar to the glucose intolerance observed in wild type pregnant females (Fig. 1B). In addition, no significant differences were found in serum insulin levels of pregnant or non-pregnant Alx3-deficient females as compared to those found in wild type animals (Supplementary Table S1). All these data indicate that Alx3-deficient non-pregnant females have mild glucose intolerance, but this is not aggravated during pregnancy, being similar to the glucose intolerance associated with pregnancy in wild type animals. Therefore, this mild alteration of glucose homeostasis cannot be expected to cause embryonic malformations.Figure 1Induction of diabetic pregnancies in wild type and Alx3-deficient mice. (A) Basal blood glucose concentrations after fasting in wild-type (n = 27) and Alx3-deficient (n = 38) female mice. **P < 0.01, Student’s t-test. (B) Glucose tolerance tests carried out in fasting wild-type (left panel, blue, n = 21) or Alx3-deficient (right panel, red, n = 19) female mice. Both non-pregnant (clear squares) and pregnant mice (solid squares) at 8.5 days post coitus were tested. *P < 0.05, **P < 0.01 relative to non pregnant wild type animals at each time point (ANOVA followed by Bonferroni transformation). No statistically significant differences were found between pregnant and non-pregnant Alx3-null females. (C) Blood glucose concentrations after fasting in wild type (white circles, n = 22) and Alx3-deficient (black circles, n = 43) female mice. Injections of streptozotocin (STZ) and subcutaneous implantation of insulin pellets (Ins) are indicated by arrows. Note that severe hyperglycaemia develops again during pregnancy up to 10.5 days of gestation. (D) Number of embryos per litter observed in non-diabetic (ND) or diabetic (D) pregnancies at 10.5 days of gestation. The numbers of litters were: Alx3 +/+, 20 (ND) and 15 (D); Alx3 −/−, 19 (ND) and 29 (D). Pregnancies without any embryos were not counted for litter size calculations. *p < 0.05; **P < 0.01, Student’s t-test. All values represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathop{+}\limits_{-}$$\end{document}+− s.e.m.