Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure

Abstract

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoenolpyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth.

Fig. 1.

Embryos developing in culture have fewer cells than those developing in vivo. (A) Mean (±SEM) blastocyst cell numbers at 96 and 116 h post-hCG after culture in T6 medium with or without protein (T6 BSA; T6 PVP) compared with blastocysts developing in vivo at 96 h post-hCG (n = 13–39 per treatment; ∗, P < 0.05 compared with in vivo group). (B) Mean (±SEM) ICM/TE ratio of blastocysts in A.

Fig. 2.

Embryo culture and transfer treatments have minimal effect on postnatal growth. Mean (±SEM) body mass and Z-score for male (A) and female (B) offspring from 1 to 27 weeks (n = 20–33 males, 20–31 females from 6–10 litters). In B, ∗ (P < 0.05) and “t” (trend; P < 0.1) denote differences between IVC-ET and IV-ET data. In Z-score plots, ∗ denotes that the entire slope is significantly different (P < 0.05) between NM and other treatment groups.

Fig. 3.

Embryo culture and transfer treatments induce elevation in postnatal SBP. Mean (±SEM) SBP of male (n = 20–31 per treatment) (A) and female (n = 19–31) (B) offspring from 6–10 litters per treatment group. Different letter denotes P ≤ 0.05 independent of maternal origin, litter size, and weight. In addition, differences at trend level (P < 0.1) exist for IVC-ET and NM (males, 21 weeks), IV-ET and NM-6 (males, 21 weeks), and IVC-ET and NM-6 (females, 15 weeks).

Fig. 4.

Embryo culture and/or transfer cause elevation in female offspring serum ACE and PEPCK activities. (A) Mean (±SEM) serum ACE activity in 16–17 male and 15–17 female offspring from 6–10 litters per treatment. (B) Mean (±SEM) liver PEPCK activity in 16–17 male and 15–18 female offspring from 8–10 litters per group. ∗, P < 0.05 compared with NM (A) or NM-6 (B) group.