Effect of in vitro fertilisation (IVF) and embryo culture duration on mouse development and postnatal health

Abstract

Since the advent of IVF (in vitro fertilisation) and assisted reproductive technologies (ART), several million babies have been born worldwide. However, reports link in vitro techniques with adverse short and long-term health outcomes. Using a mouse model, we have investigated the effect of IVF and culture on blastocyst development and cell number and on postnatal health of offspring. To explore the effect of different durations of embryo culture after IVF (as used commonly in clinical practice) and to evaluate the effect of embryo transfer itself plus the need for different controls, five treatment groups were generated as follow, each comprising 8-13 litters. NM (natural mating control, no ART treatment, non-superovulated); IV-ET-2Cell (2-cell embryos derived in vivo from superovulated (SO) mothers and immediately transferred (ET) to pseudo-pregnant recipients); IV-ET-BL (blastocysts derived in vivo from SO mothers and immediate ET); IVF-ET-2cell (2-cell embryos generated by IVF from SO mothers, short culture and ET); IVF-ET-BL (blastocysts generated by IVF from SO mothers, long culture and ET). Offspring were weighed weekly, systolic blood pressure (SBP) taken at weeks 9, 15, 21 and LIFE (average), and glucose tolerance test (GTT) carried out prior to culling for organ collection at week 27. Serum glucose, insulin concentration and the G:I ratio were calculated, with serum and lung angiotensin converting enzyme (ACE) levels determined after collection and storage of serum and lungs following culling; with random effects regression statistical analysis used to assess independence of litter size and maternal origin. IVF blastocysts after prolonged culture developed slower and comprised reduced trophectoderm and ICM cell numbers compared with in vivo generated blastocysts (P<0.05; n= 50-87 per treatment; differential nuclear labelling). Offspring from IV-ET-2Cell (n= 57), IV-ET-BL (n= 47), IVF-ET-2Cell (n= 75) and IVF-ET-BL (n= 42) groups compared with NM controls (n=80), showed increased body weight, increased SBP, impaired GGT and abnormal organ:body weight ratios in both sexes (P<0.05), independent of litter size. At weeks 15, 21 and LIFE, SBP for IVF-ET-BL males was increased compared with IV-ET-BL males (P= 0.003, 0.014 and 0.001, respectively). At weeks 15, 21 and LIFE, IVF-ET-BL males had increased SBP compared with IVF-ET-2Cell males (P=0.032, 0.034 and 0.017, respectively). In addition, offspring from the IVF-ET-BL group had a significant increase in serum and lung ACE activity compared with the NM group (P= 0.034), (P= 0.019) respectively. Offspring from IVF-ET-BL group also had a significant increase in lung ACE activity compared with IV-ET-BL group (P=0.042), although, serum ACE activity tended to be higher than IV-ET-BL, but this did not reach statistical significance (ʈ =0.070). Selected correlations show that SBP at 21 weeks in male offspring from IVF-ET-BL were positively correlated with body weight at 9 weeks (ʈ =0.051), at 15 weeks (P=0.018) and at 21 weeks (P=0.016) with R2 values of 0.046, 0.09 and 0.09 respectively. SBP at 21 weeks and LIFE were also positively correlated with lung ACE activity 0.002 and 0.009 respectively. However, glucoseconcentration 2 hours after glucose injection and the AUC (area under curve) in the male IVF-ET-BL group was reduced compared with IVF-ET-2Cell males (P= 0.03, 0.003, respectively). In males, IV-ET-2Cell, IVF-ET-2Cell and IV-ET-BL offspring all demonstrate low G:I ratios in comparison to NM mice (P=0.005, P=0.001 and P=0.038; respectively). Selected correlations demonstrate that there is a relationship between weight and AUC, in which weight is positively correlated with AUC measurements in NM (P=0.001), IV-ET-2Cell (P=0.000), IVF-ET-2Cell (P=0.046), IV-ET-BL (P=0.013) and IVF-ET-BL offspring (P=0.002), with R2 values of 0.2, 0.29, 0.13, 0.26 and 0.2, respectively. Male IVF-ET-BL heart:body weight ratio was increased and liver:body weight ratio reduced compared with IVF-ET-2Cell males (P=0.019, 0.023, respectively). No differences were evident between the four treatments groups for females. Our results suggest that reproductive treatments affect the development and potential of preimplantation embryos, influencing postnatal development and physiology compared with undisturbed reproduction. In particular, prolonged embryo culture (from 2-Cell to blastocyst), with normalised SO, IVF and ET, may adversely affect male offspring cardiovascular health, but improve the metabolic profile, compared with short culture (ET at 2-cell stage). However, female health is less sensitive.

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