Embryo cryopreservation does not induce clear-cut anomalies at detectable rates, but several mechanisms exist for nonlethal damage during the freeze-thaw process, and the risk of moderate or delayed consequences has not been extensively investigated. In a long-term study including senescence, we compared cryopreserved and control mice for several quantitative traits. Significant differences were seen in morphophysiological and behavioral features, some of them appearing in elderly subjects. Thus, apart from its immediate toxicity, embryo cryopreservation, without being severely detrimental, may have delayed effects. These results, consistent with other findings, question the neutrality of artificial reproductive technologies and draw attention to the preimplantation stages in developmental toxicology.Embryo freezing is common practice in several species including humans. This technique can be lethal to some embryos but is not considered to have any delayed effect. However, several arguments question such a viewpoint. Several targets of major importance to further development and life are present in the early mammalian embryo: the nuclear DNA, of course, and also the mitochondrial genome (1) and early processes such as those related to imprinting (2, 3), which are completed after fertilization. Freezing-thawing involves dramatic cellular and biochemical changes, such as enzyme inactivation (4-6), ionic disturbances, or attack by free radicals (7-9), that through various pathways could damage these critical components or processes. The current opinion-that embryo freezing has no late consequencesrelies on the negative results of previous experimental studies (10-15) and on common experience in cattle and humans. More recently, however, damage to the genetic material from freezing has been reported (16-18). Furthermore, in domestic species as in humans, investigations have mainly focused on patent defects at birth or in early life. However, for complex organisms in which many genetic changes, especially mitochondrial, may have only mild or delayed effects, such indicators provide only a limited assessment of the broad spectrum of anomalies that could result from mutagenic or toxic action on the early embryo.More detailed investigation and long-term follow-up are necessary to comprehensively assess embryo-freezing consequences in mammals. We report here the results of a study comparing mice derived from cryopreserved and control embryos in several morphophysiological, sensorimotor, and behavioral traits, from birth to senescence. The experiment was done on two different hybrid genotypes: C57BL6/CBA F1 (B6CBA) and C3H/DBA2 F1(C3D2).
MATERIALS AND METHODSEmbryo Collection. After superovulation (pregnant mare serum gonadotropin, 5 international units, and human chorionic gonadotropin, 5 international units, 48 hr later), 8-weekold C57BL/6JIco (B6) and C3H/OuJIco (C3) females were mated with CBA/JIco (CBA) and DBA/2JIco (D2) males, respectively. B6CBA and C3D2 embryos were collected in M2 medium (19), at the two...
