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...

The effects of urogenital infection on male fertility are controversial. The object of this study was to assess whether contact between E. coli, one of the bacteria encountered most frequently in semen cultures, and sperm was involved in decreasing motility of the sperm. Sperm from healthy donors were therefore incubated at two concentrations (1.10(7) and 4.10(7) ml-1) with bacteria (10(4) and 10(6) bacteria ml-1 respectively). Sperm motility was assessed as a function of time. The endotoxin effect was also evaluated. Aliquots of the sperm were used as controls. The motility of a population of 10(6) sperm ml-1 was reduced significantly more by the presence of 10(6) ml-1 E. coli than a sperm population four times more numerous. Since the endotoxin had no effect on sperm motility, it is possible this phenomenon is due to bacterial adherence to the sperm. From this study, it is therefore probable that the presence of E. coli in semen decreases sperm motility, but that this depends on the sperm:bacterial ratio ml semen-1.

No abstract

The effects of maternal age on the quality of offspring are well known. Those due to the father's age are less obvious, apart from the role of increasing paternal age in the onset of many dominant autosomal disorders. But an experimental model has demonstrated that, in rats, increasing paternal age, without any other anomalies, might produce a decreased learning capacity in progeny. The object of the epidemiological investigation presented here was to verify whether this effect might also occur in man. The study involved the distribution of scores obtained in psychometric tests by 18-year-old male subjects, according to their father's age at the time of their birth. This distribution indicated not only that increasing paternal age is accompanied by effects similar to those observed in animals, but also that very young paternal age was also related to these effects. Thus, the curve of such scores produced an inverted U-shape, with maximum scores obtained when the father was about thirty years of age. Maternal age did not appear to play a part in this event. These results pose the problem of identifying genetic and/or psychosocial factors which might have an impact on the quality of the conceptus.

Cryopreservation of mouse oocytes induced a high rate of atresia. Frozen oocytes observed immediately after thawing did not exhibit any alteration in the frequency of chromosomal abnormalities, aneuploidy or polyploidy. After in-vitro fertilization attempts, the cleavage rate of frozen-thawed mouse oocytes was decreased. Cytogenetical observations of inseminated eggs also confirmed this decrease in fertilization rate. First and second cleavages were delayed compared to fresh controls but subsequent development to the 4-cell stage was not altered. Freeze-thawing increased the incidence of chromosomal abnormalities in inseminated oocytes but this only concerned the frequency of triploidy and not monosomic or trisomic aneuploidy. The increase in triploidy seemed to be largely due to the presence of digynic embryos. Second polar body retention seemed to be mainly responsible for this high rate of polyploidy.

The same 15 male Wistar rats at the ages of 2.5, 6, 10, 14, 18, and 22 months were successively randomly mated with 2.5-month-old females. In a separate experiment, 15 male Wistar rats at the age of 2.5 months and 15 at the age of 23 months were simultaneously randomly mated with 2.5-month-old females. Offspring were evaluated in regard to the mean number per litter, sex ratio, frequency of gross external malformations, growth pattern, and mortality in the first 13 weeks of life and reproductive capacity at 13 weeks of age. They were also evaluated for spontaneous activity and emotionality with an open field test and for learning capacity with an avoidance conditioning test, both carried out between 10 and 13 weeks of age. Only learning capacity of the offspring, expressed in percentage of success for male or female, decreased consistently and significantly as the father's age increased. But females did not seem to be affected in the same way as males. The genetic implications are briefly discussed.

In a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen-thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing-thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0 degrees C had no effect. DMSO, used at 0 degrees C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing-thawing process.

The various nonspermatozoal cell types in the semen of 106 fertile (F) and 102 subfertile (SF) men were described and their relative proportions estimated. About 94% (F) and 90% (SF) were found to be germinal elements, among which, respectively, about 27% and 51% were spermatids, 48% and 36% residual bodies, 19.4% and 2.6% primary spermatocytes, 0.03% and 0.61% spermatogonia. The epithelial cells and blood cells represented about 6% (F) and 10% (SF) of the nonspermatozoal cells; in F men 5.3% and in SF men 9.5% were found to be polymorphonuclear leucocytes. In SF men the predominance of spermatids might be due to a particular fragility of spermiogenesis. To the three stages of spermatogenesis-the gonial multiplication, meiosis, and spermiogenesis-might correspond three specific pathologies. A pathology of the very germ cell production was thus suggested, as well as a pathology of the means by which the final product would be controlled. The Sertoli cell was supposed to be mainly involved in the latter process.