The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.

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.

More than 4.5 million children have been conceived by in vitro fertilization (IVF). Interestingly, singleton IVF offspring born at term have an increased incidence of low birth weight. The mechanism responsible for the lower birth weight is unknown, but alterations in placental function are possible. Hence, the goal of our study was to examine placental growth and function in mice generated in vivo or in vitro. To assess placental function, blastocysts were generated by IVF or produced by natural mating (control group); both IVF and control blastocysts were transferred to pseudopregnant recipients. Placental weights did not differ at embryonic d 15.5 (E15.5) but were increased at E18.5 in the IVF group (25.4%, P < 0.001) compared with control. Proliferation was increased in IVF placentae, whereas overall placental gross morphology and apoptosis were not affected. Both fetal weights (16.4% lower at E15.5 and 8.8% lower at E18.5, P < 0.05) and fetal to placental ratios were lower (P < 0.001) in the IVF compared with the control group at both time points, whereas birth weights did not differ. At E18.5, the mRNA for selected glucose, system A amino acid transporters, and imprinted genes were down-regulated in IVF placentae. GLUT3 protein level was decreased in the IVF group (P < 0.05). Importantly, intrajugular injections of (14)C-methyl-D-glucose or (14)C-MeAIB tracers (n = 6 litters per group) showed that placental transport of glucose and amino acids were 24.8% (not significant) and 58.1% (P < 0.05) lower in the IVF group. Fetal accumulation of glucose was not different, but amino acid accumulation was significantly (36 %) lower in IVF fetuses (P < 0.05). We conclude that IVF alters both fetal and placental growth and, importantly, decreases placental transport efficiency in mice conceived by IVF.

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.

The preimplantation embryo is particularly vulnerable to environmental perturbation, such that nutritional and in vitro stresses restricted exclusively to this stage may alter growth and affect long-term metabolic health. This is particularly relevant to the over 5 million children conceived by in vitro fertilization (IVF). We previously reported that even optimized IVF conditions reprogram mouse postnatal growth, fat deposition, and glucose homeostasis in a sexually dimorphic fashion. To more clearly interrogate the metabolic changes associated with IVF in adulthood, we used nontargeted mass spectrometry to globally profile adult IVF- and in vivo-conceived liver and gonadal adipose tissues. There was a sex- and tissue-specific effect of IVF on adult metabolite signatures indicative of metabolic reprogramming and oxidative stress and reflective of the observed phenotypes. Additionally, we observed a striking effect of IVF on adult sexual dimorphism. Male-female differences in metabolite concentration were exaggerated in hepatic IVF tissue and significantly reduced in IVF adipose tissue, with the majority of changes affecting amino acid and lipid metabolites. We also observed female-specific changes in markers of oxidative stress and adipogenesis, including reduced glutathione, cysteine glutathione disulfide, ophthalmate, urate, and corticosterone. In summary, embryo manipulation and early developmental experiences can affect adult patterns of sexual dimorphism and metabolic physiology.

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Stressful environmental exposures incurred early in development can affect postnatal metabolic health and susceptibility to non-communicable diseases in adulthood, although the molecular mechanisms by which this occurs have yet to be elucidated. Here we use a mouse model to investigate how assorted in vitro exposures restricted exclusively to the preimplantation period affect transcription both acutely in embryos and long-term in subsequent offspring adult tissues, to determine if reliable transcriptional markers of in vitro stress are present at specific developmental time points and throughout development. Each in vitro fertilization or embryo culture environment led to a specific and unique blastocyst transcriptional profile, but we identified a common 18-gene and 9-pathway signature of preimplantation embryo manipulation that was present in all in vitro embryos irrespective of culture condition or method of fertilization. This fingerprint did not persist throughout development and there was no clear transcriptional cohesion between adult IVF offspring tissues or compared to their preceding embryos, indicating a tissue-specific impact of in vitro stress on gene expression. However, the transcriptional changes present in each IVF tissue were targeted by the same upstream transcriptional regulators, which provide insight as to how acute transcriptional responses to stressful environmental exposures might be preserved throughout development to influence adult gene expression.

Prenatal development is highly plastic and readily influenced by the environment. Adverse conditions have been shown to alter organ development and predispose offspring to chronic diseases, including diabetes and hypertension. Notably, it appears that the changes in glucocorticoid hormones or glucocorticoid receptor (GR) levels in peripheral tissues could play a role in the development of chronic diseases. We have previously demonstrated that in vitro fertilization (IVF) and preimplantation embryo culture is associated with growth alterations and glucose intolerance in mice. However, it is unknown if GR signaling is affected in adult IVF offspring. Here we show that GR expression is increased in inbred (C57Bl6/J) and outbred (CF-1× B6D2F1/J) blastocysts following in vitro culture and elevated levels are also present in the adipose tissue of adult male mice. Importantly, genes involved in lipolysis and triglyceride synthesis and responsive to GR were also increased in adipose tissue, indicating that increased GR activates downstream gene pathways. The promoter region of GR, previously reported to be epigenetically modified by perinatal manipulation, showed no changes in DNA methylation status. Our findings demonstrate that IVF results in a long-term change in GR gene expression in a sex- and tissue-specific manner. These changes in adipose tissues may well contribute to the metabolic phenotype in mice conceived by IVF.