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Reproduction (2005) 130 599-601
DOI: 10.1530/rep.1.00872
Copyright © 2005 Society for Reproduction and Fertility
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BRIEF COMMUNICATION

Embryo culture does not affect the longevity of offspring in mice

Joshua Sommovilla1, Warren B Bilker2, Ted Abel1 and Richard M Schultz1

1 Department of Biology and 2 Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA 19104, USA

Correspondence should be addressed to R M Schultz; Email: rschultz{at}sas.upenn.edu


   Abstract
Top
 Abstract
Longevity and embryo culture
 Acknowledgements
 References
 
The oldest assisted reproductive technologies (ART)-conceived child is only 27 years old. Thus, the effects of ART on longevity are unknown, and it will be many years before this can be assessed in humans. We recently reported that culturing preimplantation mouse embryos under suboptimal conditions results in differences in how the offspring perform in behavioral assays that reflect anxiety (elevated zero maze) and spatial memory (Morris hidden water maze; Ecker et al. 2004). Here we monitored the mice generated in our previous study and found no difference in their longevity.


   Longevity and embryo culture
Top
 Abstract
 Longevity and embryo culture
Acknowledgements
 References
 
Since the birth of Louise Brown in 1978, use of assisted reproductive technologies (ART) to treat human infertility has grown at a rapid rate, accelerating with the implementation of intracytoplasmic sperm injection (ICSI) in the early 90’s such that in many developed countries ART accounts for 1–3% of births (Maher 2005). Culture of preimplantation embryos prior to embryo transfer is typically an intrinsic part of ART. Culture, however, is known to affect both embryo metabolism (Gardner et al. 2000) and the global pattern of gene expression (Rinaudo & Schultz 2004), including imprinted genes (Doherty et al. 2000, Khosla et al. 2001, Mann et al. 2004). Regarding imprinting, culture can lead to bi-allelic H19 expression that is linked with loss of DNA methylation of the paternal allele in the differentially methylated region (Mann et al. 2004); DNA methylation of this region is essential for repression of the paternal allele. Loss of imprinting can lead to specific syndromes in humans, including Angelman and Beckwith–Weidemann syndromes. Of particular note is that a significantly higher incidence of these syndromes is observed in ART-conceived children, and in most instances this is due to loss of DNA methylation in critical regulatory sequences (Maher 2005), i.e. the syndrome is due to an epigenetic change. In addition, there are reports of an increased incidence of cerebral palsy (Strömberg et al. 2002) and low birth-weight (Schieve et al. 2002) in ART-conceived children. Because the effects of ART on longevity in humans will not be known for many years, we monitored the mice generated in our previous study to ascertain if there were any significant differences in their longevity. Our analysis, which did not find any significant difference in lifespan after culture (Fig. 1A–CGo), is described below.



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  		Figure 1 Percentage survival in offspring following culture in vitro or development in vivo. A; survival of all animals, B; survival as a function of sex for males, C; survival as a function of sex for females, D; survival as a function of sex. See text for discussion of statistical analysis.

 
We compared the lifetimes of mice that were from two difference sources, namely, those that developed in vivo and those that developed in vitro in either KSOM or Whitten’s medium. Because there are sex differences in mouse lifetimes (Fig. 1DGo), an adjustment was made for sex. In addition, in the original experimental design three different genotypes were generated due to mating females with either wild-type males or males harboring either a Teto or Line30 transgene. See Ecker et al.(2004) for further discussion on the rationale for this experimental design. Adjustment for genotype was also considered. In addition, interaction effects on lifetime between sex, genotype, and in vivo/in vitro were considered. Although the complete natural lifetime was observed for most mice, there were some cases of incomplete lifetimes and some mice required euthanasia. The lifetimes were compared using the Cox proportional hazards regression model, which accommodates such incomplete or censored observations (Cox 1972).

There were a total of 290 mice. Complete natural lifetime in 237 mice (53 in vivo, 88 KSOM, 96 Whitten’s). Death time was known within an interval but the death date was not precisely known for 40 mice (10 in vivo, 15 KSOM, 15 Whitten’s) in which case the interval midpoint was used as time of death. 2 mice were euthanized after developing lethal conditions (0 in vivo, 1 KSOM, 1 Whitten’s; euthanization date taken as time of death). 9 mice were euthanized with non-lethal ailments such as fighting wounds (1 in vivo, 5 KSOM, 3 Whitten’s; euthanization date taken as time of death), and 2 mice were still alive at the time of this analysis (0 in vivo, 0 KSOM, 2 Whitten’s; the last observed time was used as the censored time).

The Cox regression model included in vivo/in vitro and sex as predictors of lifetime. The Cox model results are in terms of hazard ratios or instantaneous relative risk. For example, the hazard ratio (HR) for the in vivo variable is interpreted as the instantaneous relative risk of death at any time for an in vitro-derived mouse compared with an in vivo-derived mouse, where a significant hazard ratio less than 1 would indicate that at any point in time the risk of death for an in vivo-derived mouse is less than for an in vitro-derived mouse, indicating a longer lifetime for an in vivo-derived mouse.

The result indicates that in vivo status does not impact the lifetimes (HR = 0.916, P = 0.541, 95% confidence interval 0.692–1.213), while sex does impact the lifetimes (HR = 0.677, P = 0.001, 95% confidence interval 0.535–0.857), with males having longer lifetimes. Thus, after adjusting for sex, there is no difference in lifetimes between in vivo and in vitro-derived mice. An additional model was fit to consider the three mouse genotypes included in the study. Including genotype in the above Cox model does not alter the conclusions. The result indicates that in vivo status does not impact on lifetime (HR = 0.815, P = 0.176, 95% confidence interval 0.606–1.096), while the male sex has longer lifetimes (HR = 0.652, P < 0.001, 95% confidence interval 0.513–0.827) compared to females. Genotype does impact on lifetime (P = 0.021), with wild type and Teto lifetimes not significantly different to each other (P = 0.801), but line30 mice having shorter lifetimes compared to both wild type (HR = 1.442, P = 0.020, 95% confidence interval 1.059–1.966) and to Teto mice (HR = 1.495, P = 0.010, 95% confidence interval 1.103–2.027). The proportional hazards assumption of the Cox model was assessed both graphically and via a hypothesis test based on Schoenfeld residuals (Schonfeld 1982), and both indicate that the required assumption is satisfied for these models. Each of the potential two-way interactions between in vivo/in vitro, genotype, and sex were all considered for inclusion in the model. None of these interactions were significant and there was no impact on the non-significant result obtained for the in vivo/in vitro status.

In summary, results using a mouse model in which embryo culture had long-term effects on behavior in the offspring had no effect on longevity. These results suggest that although there is a significant increase in risk associated with ART, a detrimental effect on longevity is unlikely to be one of them.


   Acknowledgements
Top
 Abstract
 Longevity and embryo culture
 Acknowledgements
References
 
This research was supported by a grant from the NIH (U01 HD 44575 to RM Schultz and T Abel as part of the NICHD Cooperative Program on Female Health and Egg Quality and P50 MH 64045 to RE Gur). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 14 June 2005
First decision 6 July 2005
Revised manuscript received 6 July 2005
Accepted 22 August 2005


   References
Top
 Abstract
 Longevity and embryo culture
 Acknowledgements
 References
 

Cox DR 1972 Regression models and life-tables. Journal of the Royal Statistical Society Series B 34 187–202.

Doherty AS, Mann MR, Tremblay KD, Bartolomei MS & Schultz RM 2000 Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biology of Reproduction 62 1526–1535.[Abstract/Free Full Text]

Ecker DJ, Stein P, Xu Z, Williams CJ, Kopf GS, Bilker WB, Abel T & Schultz RM 2004 Long-term effects of culture of preimplantation mouse embryos on behavior. PNAS 101 1595–1600.[Abstract/Free Full Text]

Gardner DK, Pool TB & Lane M 2000 Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and viability. Seminars in Reproductive Medicine 18 205–218.[CrossRef][ISI][Medline]

Khosla S, Dean W, Brown D, Reik W & Feil R 2001 Culture of Preimplantation Mouse Embryos Affects Fetal Development and the Expression of Imprinted Genes. Biology of Reproduction 64 918–926.[Abstract/Free Full Text]

Maher ER 2005 Imprinting and assisted reproductive technology. Human Molecular Genetics 14 Supplement 1 R133–R138.[Abstract/Free Full Text]

Mann MR, Lee SS, Doherty AS, Verona RI, Nolen LD, Schultz RM & Bartolomei MS 2004 Selective loss of imprinting in the placenta following preimplantation development in culture. Development 131 3727–3735.[Abstract/Free Full Text]

Rinaudo P & Schultz RM 2004 Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryo. Reproduction 128 301–311.[Abstract/Free Full Text]

Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G & Wilcox LS 2002 Low and very low birth weight in infants conceived with use of assisted reproductive technology. New England Journal of Medicine 346 731–737.[Abstract/Free Full Text]

Schonfeld D 1982 Partial residuals for the proportional hazards regression model. Biometrika 69 239–241.[Abstract/Free Full Text]

Strömberg B, Dahlquist G, Ericson A, Finnström O, Köster M & Stjernqvist K 2002 Neurological sequelae in children born after in vitro fertilisation: a population-based study. Lancet 359 461–465.[CrossRef][ISI][Medline]


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