In vitro fertilization affects growth and glucose metabolism in a sex-specific manner in an outbred mouse model

Abstract

The preimplantation period is a time of reprogramming that may be vulnerable to disruption. This question has wide clinical relevance since the number of children conceived by in vitro fertilization (IVF) is rising. To examine this question, outbred mice (CF1 × B6D2F1) conceived by IVF and cultured using Whitten medium and 20% O2 (IVFWM group, less optimal) or K simplex optimized medium with amino acids and 5% O2 (IVFKAA group, more optimal and similar to conditions used in human IVF) were studied postnatally. We found that flushed blastocysts transferred to recipient mice provided the best control group (FB group), as this accounted for the effects of superovulation, embryo transfer, and litter size. We observed that many physiological parameters were normal. Reassuringly, IVFKAA offspring did not differ significantly from FB offspring. However, male IVFWM mice (but not females) were larger during the first 19 wk of life and exhibited glucose intolerance. Male IVFWM mice also showed enlarged left heart despite normal blood pressure. Expression of candidate imprinted genes (H19, Igf2, and Slc38a4) in multiple adult tissues did not show differences among the groups; only Slc38a4 was down-regulated following IVF (in both culture conditions) in female adipose tissue. These studies demonstrate that adult metabolism is affected by the type of conditions encountered during the preimplantation stage. Further, the postnatal growth trajectory and glucose homeostasis following ex vivo manipulation may be sexual dimorphic. Future work on the long-term effects of IVF offspring should focus on glucose metabolism and the cardiovascular system.

Keywords: ART; DOHaD; embryos.

FIG. 1

Growth curves. Female (A, B) and male (C, D) IVF_WM and IVF_KAA mice were compared separately to FB control mice. The IVF_WM males (C) were significantly larger (*) than their FB counterparts for most of their growth. Performing the GTT (arrows) appeared to temporarily disrupt the growth of the mice. N for each time point are given in Supplemental Table S2. Values are means ± SD.

FIG. 2

Glucose tolerance test. Female IVF mice (A) had a normal glucose tolerance as evidenced by glucose levels over time and by the calculation of the AUC (C). Male IVF_WM mice, however, showed glucose intolerance, maintaining significantly higher levels of serum glucose (*) after intraperitoneal glucose injection (B). This was also reflected in the significantly higher AUC for male IVF_WM mice (D). The insulinogenic index (E) was significantly lower (#) in IVF mice. The insulin resistance index (HOMA-IR; F) for males was not different between FB and IVF_WM groups at either 19 or 40 wk (n = 9; three litters; same mice at both time points) but increased significantly (Δ) with time. Values are means ± SD.

FIG. 3

Hyperinsulinemic-euglycemic clamp. A) Glucose levels were maintained in both FB and IVF_WM males at approximately 100 ng/dl at clamp. B) Glucose infusion rate was similar in both groups. C) Total glucose flux at baseline was not statistically different between IVF_WM mice and control FB mice, but at clamp (the average of the 100- and 120-min values), it was significantly lower in IVF_WM mice (Supplemental Table S3; n = 9–11 per group from three litters per treatment). D) The endogenous glucose production rate was similar in both groups at both baseline and clamp. E) Glucose uptake measured in various organs was also not different. Values are means ± SD.