title: Advanced maternal age causes adverse programming of mouse blastocysts leading to altered growth and impaired cardiometabolic health in post-natal life.
abstract: Does advanced maternal age (AMA) in mice affect cardiometabolic health during post-natal life in offspring derived from an assisted reproduction technology (ART) procedure?
  
  
  
  Offspring derived from blastocysts collected from aged female mice displayed impaired body weight gain, blood pressure, glucose metabolism and organ allometry during post-natal life compared with offspring derived from blastocysts from young females; since all blastocysts were transferred to normalized young mothers, this effect is independent of maternal pregnancy conditions.
  
  
  
  Although studies in mice have shown that AMA can affect body weight and behaviour of offspring derived from natural reproduction, data on the effects of AMA on offspring cardiometabolic health during post-natal development are not available. Given the increasing use of ART to alleviate infertility in women of AMA, it is pivotal to develop ART-AMA models addressing the effects of maternal aging on offspring health.
  
  
  
  Blastocysts from old (34-39 weeks) or young (8-9 weeks) C57BL/6 females mated with young CBA males (13-15 weeks) were either subjected to differential cell staining (inner cell mass and trophectoderm) or underwent embryo transfer (ET) into young MF1 surrogates (8-9 weeks) to produce young (Young-ET, 9 litters) and old (Old-ET, 10 litters) embryo-derived offspring. Offspring health monitoring was carried out for 30 weeks.
  
  
  
  All animals were fed with standard chow. Blood pressure was measured at post-natal Weeks 9, 15 and 21, and at post-natal Week 30 a glucose tolerance test (GTT) was performed. Two days after the GTT mice were killed for organ allometry. Blastocyst cell allocation variables were evaluated by T-test and developmental data were analysed with a multilevel random effects regression model.
  
  
  
  The total number of cells in blastocysts from aged mice was decreased (P < 0.05) relative to young mice due to a lower number of cells in the trophectoderm (mean ± SEM: 34.5 ± 2.1 versus 29.6 ± 1.0). Weekly body weight did not differ in male offspring, but an increase in body weight from Week 13 onwards was observed in Old-ET females (final body weight at post-natal Week 30: 38.5 ± 0.8 versus 33.4 ± 0.8 g, P < 0.05). Blood pressure was increased in Old-ET offspring at Weeks 9-15 in males (Week 9: 108.5 ± 3.13 versus 100.8 ± 1.5 mmHg, Week 15: 112.9 ± 3.2 versus 103.4 ± 2.1 mmHg) and Week 15 in females (115.9 ± 3.7 versus 102.8 ± 0.7 mmHg; all P < 0.05 versus Young-ET). The GTT results and organ allometry were not affected in male offspring. In contrast, Old-ET females displayed a greater (P < 0.05) peak glucose concentration at 30 min during the GTT (21.1 ± 0.4 versus 17.8 ± 1.16 mmol/l) and their spleen weight (88.2 ± 2.6 ± 105.1 ± 4.6 mg) and several organ:body weight ratios (g/g × 10(3)) were decreased (P < 0.05 versus Young-ET), including the heart (3.7 ± 0.06 versus 4.4 ± 0.08), lungs (4.4 ± 0.1 versus 5.0 ± 0.1), spleen (2.4 ± 0.06 versus 3.2 ± 0.1) and liver (36.4 ± 0.6 versus 39.1 ± 0.9).
  
  
  
  Results from experimental animal models cannot be extrapolated to humans. Nevertheless, they are valuable to develop conceptual models that can produce hypotheses for eventual testing in the target species (i.e. humans).
  
  
  
  Our data show that offspring from mouse embryos from aged mothers can develop altered phenotypes during post-natal development compared with embryos from young mothers. Because all embryos were transferred into young mothers for the duration of pregnancy to normalize the maternal in vivo environment, our findings indicate that adverse programming via AMA is already established at the blastocyst stage. Whilst human embryos display increased aneuploidy compared with mouse, we believe our data have implications for women of AMA undergoing assisted reproduction, including surrogacy programmes.
  
  
  
  This work was supported through the European Union FP7-CP-FP Epihealth programme (278418) to T.P.F. and the BBSRC (BB/F007450/1) to T.P.F. The authors have no conflicts of interest to declare.
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO16 6YD, UK School of Agriculture, Food & Rural Development, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK.
authors.forename: M A
authors.initials: MA
authors.lastname: Velazquez
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO16 6YD, UK.
authors.forename: C G C
authors.initials: CG
authors.lastname: Smith
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO16 6YD, UK.
authors.forename: N R
authors.initials: NR
authors.lastname: Smyth
authors.affiliation: MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton SO16 6YD, UK.
authors.forename: C
authors.initials: C
authors.lastname: Osmond
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton SO16 6YD, UK t.p.fleming@soton.ac.uk.
authors.forename: T P
authors.initials: TP
authors.lastname: Fleming
mesh_terms: D000367:Age Factors; D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D001794:Blood Pressure / Q000502:physiology*; D001835:Body Weight / Q000502:physiology*; D005260:Female; D005951:Glucose Tolerance Test; D008297:Male; D008423:Maternal Age; D051379:Mice; D027724:Reproductive Techniques, Assisted
keywords: blastocysts; developmental programming; gender effects; maternal aging; offspring health