The effect of vitrification on embryo development and subsequently postnatal health using a mouse model

Abstract

Animal models have shown that vitrification impairs ultrastructure and developmental potential of the oocyte, embryo survival rate, pregnancy rate and results in low birth weight of offspring but any long term effects on offspring are still unknown. In this study, embryos were vitrified at the 8-cell stage and kept in LN2. The first experiment investigated the effect of vitrification on numbers of surviving cells (comparing vitrified and non-vitrified embryos). The blastocysts developed from each group were analysed according to cell number and allocation to trophectoderm or inner cell mass (ICM). In the second experiment, embryos were warmed and cultured until they formed blastocysts before being transferred to foster mothers. Offspring were weighed weekly, systolic blood pressure (SBP) was taken at weeks 9, 15, 21, glucose tolerance tests (GTT), serum glucose and insulin concentration analyses (subset experiment) were carried out prior to culling for organ collection at week 27. Three different treatment groups were studied (10 litters in each group); 1) control (non-ART treatment) 2) embryos cultured from 8-cell to blastocyst stage followed by embryo transfer (ET) to pseudo-pregnant mothers; non-vitrified transfer(NVT) and 3) vitrified 8-cell embryos cultured to blastocyst followed by ET; vitrified transfer(VT).

In the first experiment, differential nuclear labelling of blastocysts showed vitrification significantly (p<0.05) reduced both trophectoderm and ICM cell numbers as compared to control (n=78 to 95 blastocysts per treatment). In the second experiment, in comparison to control (n=80), offspring body weight for both sexes showed a significant increase (P<0.05) in NVT (n=57) and VT (n=50) groups. Both male and female offspring demonstrated a significant increase of SBP in weeks 15, 21 and LIFE (average across all 3 time points) besides having a significantly higher glucose level as compared to control. Analysis of organ to body weight ratio found that liver, lung and heart were significantly heavier in both treatment groups for both male and female. A gender specific effect was only observed in a subset experiment of serum insulin concentration and G:I ratio in which the VT group (n=7) had significantly higher levels than the control (n=9), particularly in female offspring. However, there was no significant difference between the NVT and VT groups in terms of all parameters mentioned above. In addition, perinatal weight at week 3 (W3) is correlated significantly positive with SBP LIFE in female from the VT group (r2 = 0.1164). There is also a positive correlation between W3 and GTT AUC in male from the VT group. The subset sample showed that there is a significant positive correlation between W3 and serum insulin concentration and a significant negative correlation between W3 and G:I ratio in the VT female offspring. These results indicate that VT offspring, especially female are more prone to elevate blood pressure or hypertension if they are overweight. In addition, overweight females from VT also result in a higher serum insulin concentration that suggests insulin resistance. Interestingly, male offspring from VT group showed that being overweight causes high glucose levels, which may lead to diabetes.

In conclusion, the results suggest that even though vitrification alters blastocyst development, it does not per se change postnatal body weight, blood pressure and glucose tolerance in mice, while in vitro culture or embryonic transfer procedures may alter these parameters. Vitrification however, results in a correlation between being overweight and hypertension as well as insulin resistance, particularly in VT females. Further studies on key regulators such as ACE that regulate blood pressure and PEPCK, regulator of gluconeogenesis would further explain the link between vitrification and low surviving cell number and postnatal health effects including the observed significant correlations. The European Research Council (FP7-EPIHEALTH) and MARA, Malaysia, supported this work.

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