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.

In Western society, couples increasingly delay parenthood until later in life. Overall, studies have focused on the reproductive performance of older parents or the impact of advanced maternal age on pregnancy outcomes, but few studies have examined how advanced paternal age (APA) affects offspring health. The aim of this study was to investigate the impact of increasing paternal age on offspring reproductive performance and long-term metabolic health in a mouse model. Here, the same adult B6D2F1/J male mice were mated at 4, 12, and 18 months of age with 6- to 10-week-old naturally cycling CF1 females to generate 3 offspring cohorts conceived at increasing paternal ages PA4, PA12, and PA18. The offspring resulting from mating the same fathers at different ages (n = 20 per age; 10 males and 10 females) were maintained up to 20 weeks of age and morphometric parameters, growth curve, and glucose tolerance were measured. We found that increasing paternal age was associated with a trend toward longer time to conception. Litter sizes were not significantly different. Reassuringly, metabolic parameters and growth curve were not different in the 3 cohorts of offspring. Most importantly, increased paternal age (PA4 vs PA18) was associated with a statistically significant decrease in sperm concentration, sperm motility, and anogenital distance in offspring. These changes raise concerns about the potential impact of APA on the reproductive fitness in males of the next generation.

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.