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Human Reproduction Unit, Departments of Physiology,3 and Medicine,4 University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
ABSTRACT
Perturbations of the development of preimplantation embryos may have long-term consequences for the health of progeny. There are no standardized methods for assessing such risks. The OECD/OCDE 416 Guideline for Testing of Chemicals (Two-Generation Reproduction Toxicity Study) is a standardized assay for detecting potential toxic effects of chemicals. The present study assessed the utility of this guideline for identifying long-term consequences of perturbing preimplantation development. Extended culturing of mammalian zygotes commonly results in retarded preimplantation development. Mouse zygotes were cultured in vitro for 96 h until the blastocyst stage (cultured blastocysts) or blastocysts were collected from the Day-3.5 uterus (in vivo blastocysts). The resulting blastocysts were transferred to the uteri of pseudopregnant recipients (P generation). Progeny from both treatments were mated for a further two generations (F1 and F2 generations). There was no effect of treatment group on gross fertility across the generations tested. Progeny of the cultured blastocysts had lower body weights to the time of weaning compared to in vivo blastocysts in the P and F1 generations, but not in the F2 generation. At maturity, there was no effect of treatment group on body weight, although thyroid weight was higher in the in vivo blastocyst group in the P generation, while the brain, pituitary, and kidneys were larger in the progeny of the cultured blastocysts of the F1 generation. The OECD/OCDE 416 assessment may have a role as a standardized test for the assessment of the biological consequences of perturbing the growth environment of the preimplantation embryo. Embryo culture influenced the somatometric parameters of the resulting progeny, some of which were maintained across a generation.
assisted reproductive technology, embryo transfer, environment, epigenetics, in vitro fertilization, toxicity testing
Several standard methods exist to assess the long-term consequences of environmental perturbation of germ cell or fetal development. The possibility that perturbation of the development of the preimplantation embryo has long-term consequences has not been subjected to a thorough systematic study. Such investigations have been limited by the absence of standardized tools of assessment. Several lines of evidence indicate that perturbations of the preimplantation embryo may have long-term consequences. For example, feeding female rats a low protein diet during only the preimplantation period of pregnancy development results in blastocysts with fewer cells, with the resulting progeny showing alterations in birth weight, postnatal growth rate, hypertension, and organ:body-weight ratios [1].
Most forms of assisted reproductive technology (ART) involve the culture of the preimplantation embryo in artificial media for varying periods of time. Culturing of embryos in vitro commonly results in reduced numbers of cells in the resulting blastocysts. Population studies have shown that human infants born from ART are more likely to be small or very small compared to naturally conceived neonates, even when differences in the incidence of multiple pregnancies are taken into account [2]. There are reports of an increased incidence of congenital abnormalities for infants conceived by intracytoplasmic sperm injection (ICSI) [3, 4], and an unconfirmed report of increased incidence of a pediatric cancer [5]. In contrast, other studies have found no such risk of cancer [6]. Some studies have found an increased incidence of syndromes caused by errors in gene imprinting [7–11], a finding consistent with evidence from animal studies that embryo manipulation in vitro can influence epigenetic regulation of gene expression [12–15]. Other studies have found no difference in the psychological status at school age of children conceived by in vitro fertilization (IVF), compared to a normally conceived cohort [16]. There are conflicting reports of neurological defects, such as cerebral palsy, in IVF progeny, with some reporting an increased incidence [17], whereas others do not [18].
Within the broader area of toxicological investigation, the OECD/OCDE 416 Guideline for Testing of Chemicals (Two-Generation Reproduction Toxicity Study) is widely used for assessing gross toxicity associated with new chemical products or formulations [19]. The test is an exploratory tool for gaining general information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems, as well as the growth and development of the offspring. It also examines these parameters in the F1 and F2 generations. This test is accepted by many regulatory authorities as preliminary evidence of safety. The aim of the current study was to assess whether a modified form of the OECD 416 is informative as a means of assessing the biological impact of perturbing the growth environment of the preimplantation embryo. Since embryo culturing is a widely used medical treatment that is known to perturb the preimplantation embryo, we have chosen to examine the effects of embryo culture in this model.
The OECD 416 guideline is designed to test the effects of administered chemicals on animals from the time of weaning until sexual maturity. Central to most ART procedures is the need to culture zygotes within artificially designed culture media for varying periods. The OECD 416 was modified so that embryos cultured from the zygote stage to the blastocyst stage (96-h culture) could be compared with morphologically equivalent blastocyst stage embryos collected directly from the uterus without culture. Both groups of blastocysts were then transferred to the uteri of naïve recipients. The progeny from these two treatments were then mated naturally for two generations, and the fertility and well-being of the animals were monitored.
The present study shows that culturing of zygotes has no impact on the gross reproductive performance or fertility of mice over three generations. There was a small but significant reduction in growth rate to weaning in the cultured blastocyst group, which persisted into the first generation, although there was no effect on the weights of mature animals. However, in mature animals, treatment influenced the weights of the brain, pituitary, thyroid, and kidney. These results suggest that modified forms of the OECD 416 test may provide a basis for standardized investigation of the consequences of perturbing the growth of the preimplantation embryo on progeny and the transgenerational consequences of such perturbations.
The present study used the OECD/OCDE 416 Guideline for Testing of Chemicals (Two-Generation Reproduction Toxicity Study) as a model [19]. Because this guideline requires strains of high fecundity, hybrid (C57BL/6J x CBA/He; B6CBF1) mice were used as embryo donors. We used the Sydney IVF suite of culture media (Cook Australia P/L, Brisbane, QLD, Australia).
The use of animals was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose and was approved by the Institutional Animal Care and Ethics Committee. All animals were housed and bred in the Gore Hill Research Laboratory (St. Leonards, NSW, Australia). All animals were maintained under a 12L:12D cycle and had access to food and water ad libitum. Food was standard rodent chow provided in pellet form. Each breeding pair was housed in a plastic box (15 x 15 x 35 cm), with fresh sterile sawdust bedding that was changed twice weekly.
Six-week-old females had ovulation induced by i.p. injection of 5 IU eCG (Folligon; Intervet International, Boxmeer, The Netherlands) followed 48 h later by 5 IU hCG (Chorulon; Intervet). Females were paired with males overnight. Day 0.5 of pregnancy was confirmed by the presence of a copulation plug the following morning. Animals were checked daily for health status.
Embryos for transfer were collected by dissecting the oviduct or uterus from females that had been subjected to ovulation induction and successful mating. Zygotes were collected by tearing the infundibulum of the oviduct at about 1300 h on Day 0.5 of pregnancy in 0.5 ml of collection buffer (K-SIWB; Cook Australia), and blastocyst stage embryos were collected by flushing the lumen of the uterine horns of Day-3.5 pregnant females with 
1 ml K-SIWB. Zygotes were briefly exposed to Sydney IVF hyaluronidase (K-SIHY) medium to disperse any remaining cumulus cells. They were then either subjected to culture (zygotes) or immediately transferred to the uteri of recipient females (blastocysts).
Culture plates (60-well, 10 µl/well; Nunc, Naperville, IL) were used for all culture procedures. Ten-microliter drops of the test culture media were placed into each well of a plate, which were then overlaid with heavy paraffin oil (embryo culture grade; Sigma Chemical Co., St. Louis, MO) to a depth of 2 mm. Each plate was equilibrated in the culture incubator for at least 4 h prior to use. Embryos were cultured at a density of ten embryos per 10 µl of medium.
Zygotes were cultured for 48 h in Sydney IVF Cleavage Medium (K-SICM), and for an additional 48 h in Sydney IVF Blastocyst Medium (K-SIBM). Morphologically normal blastocysts resulting from culture were collected for embryo transfer.
Surgical Uterine Embryo Transfer
Embryo transfer was performed as previously described [20]. Pseudopregnancy was induced by mating with vasectomized males that had been confirmed as being infertile. Females of the Quackenbush strain were used, because their white coat fur allows the colored coat of B6CBF1 progeny to be readily identified as the unique progeny of embryo transfer. Pseudopregnant mice on Day 2.5 were anesthetized with an i.p. injection of 2.5% (w/v) avertin. Following incision through the skin and peritoneum, the ovarian fat pad was gently pulled out to expose the ovary, oviduct, and uterus. A total of 16 blastocysts were transferred to each recipient (eight to each uterine horn). Embryos for transfer were placed into a glass capillary (100-µm internal diameter), which was inserted into the lumen of the uterus, following puncture of the uterine wall with a sterile single-use 29G needle. The embryos were expelled into the uterus. Following embryo transfer, the incisions into the peritoneum and skin were closed separately with 5–0 silk sutures. The mouse was kept on a warm tray until it regained consciousness. The progeny born from embryo transfer are referred to as the P generation.
Male and female P generation progeny were selected randomly. They were housed together as pairs and the incidence of normal mating was recorded. Twenty mating pairs were created for each treatment group, thereby ensuring that no sibling mating occurred.
Females were observed daily for evidence of mating. Once successful mating was recorded, the males were removed from the females. Mated females were housed individually in maternity cages. If mating had not occurred after 1 wk, a new male was introduced to the female, and the first male was introduced to a new naïve female. The progeny that resulted from this cross formed the F1 generation.
From each treatment group, 20 F1 generation males and females were selected randomly at weaning to act as F1 parents (while ensuring that no sibling mating occurred). The mice selected as F1 parents were mated (as described above) and the resulting progeny formed the F2 generation. The P and F1 generation males were killed either after their partners had delivered a litter or 2 wk after unsuccessful mating (age: 152 ± 29.8 days, mean ± SD). The females were killed after weaning of their litters (age: 162 ± 25.8 days). At the time of death, body weight and the weight of the following organs of the parental animals were determined: brain, pituitary, thyroid, liver, spleen, adrenals, kidneys, and reproductive organs (uterus, ovaries, and vagina of the females; and testes, epididymides, prostate, and seminal vesicles of the males). In the case of paired organs, each member was weighed separately and the results shown are the average weight of each member of the pair. All necroscopies, organ collection, and measurements were performed by the same experienced operator throughout the study.
Each litter that resulted from these matings was examined on the day of delivery (lactation Day 0). Pups were weighed individually at birth (lactation Day 0) and on Days 4, 7, 14, and 21 of lactation. Pups were weaned on Day 21.
Quantitative results were analyzed using the SPSS for Windows version 11.5.0 program (SPSS Inc., Chicago, IL). Normality of the data was tested by the Kolmogorov-Smirnov statistic and equality of variance was assessed using the Levene Test. Data were analyzed using univariate or multivariate ANOVA within the general linear model. Differences in main factor effects were analyzed by simple contrast analysis within the general linear model. The effects of treatment and generation on birth weight and growth rate up to the time of weaning were assessed by repeated measures ANOVA. The data were clustered for effect of litter identity. For all ANOVA tests, a range of covariates was entered into the statistical models, as described in the relevant section of Results. The analyses comparing results between generations involved multiple independent tests and outcomes and were therefore subjected to Bonferroni correction [21]. The P values shown are those following correction. Success of embryo transfer or mating was analyzed by the Fisher exact test. Comparison of two parameters was by t-test. For all analyses, differences with P < 0.05 were taken to be statistically significant.
The OECD/OCDE 416 Guideline for Testing of Chemicals is designed to assess the performance of the male and female reproductive systems, as well as the growth and development of the offspring over several generations. The guideline is designed for testing the effects of administered chemicals on animals. Because it is not directly applicable to the testing of embryo culture conditions, the guideline has been modified. Culturing of zygotes in a synthetic culture medium is common to all forms of modern ART. The present study assessed the effects of zygote culture to the blastocyst stage by comparing the progeny that resulted from embryo transfer of cultured embryos with transfer of similar stage blastocysts collected directly from the reproductive tract and transferred to recipients without culturing. Zygotes cultured in vitro develop at a slower rate than those in the reproductive tract. In our model, we matched embryos according to developmental stage rather then chronological age. Thus, transfer of zona pellucida-intact blastocyst stage embryos was performed for both groups. The progeny produced by these procedures were mated over two subsequent generations.
Morphologically normal blastocysts that resulted from the culturing of zygotes or that were collected directly from the uteri of recipients were surgically transferred to the uteri of pseudopregnant recipient females. Embryo transfer of either fresh blastocysts or those cultured from the zygote stage resulted in equally high rates of ongoing successful pregnancies (P > 0.05; Fig. 1A). There were no differences in average birth weight (P > 0.05; Fig. 1B), litter size (P > 0.05; Fig. 1C) or numbers of pups surviving to weaning (P > 0.05; Fig. 1D) between the two treatment groups. Upon achieving sexual maturity, the progeny of both groups were mated naturally for a further two generations. The proportion of animals that successfully mated was not different (P > 0.05) between the groups in the P generation (cultured blastocysts, 95%; in vivo blastocysts, 100%) or F1 generation (cultured blastocysts, 100%; in vivo blastocysts, 90%). All animals (male and female) that were unsuccessful in mating with their initial partners were successful when introduced to a new partner. There was no difference in each generation (P > 0.05) in the number that had successful litters (P: zygote, 95%, and blastocyst, 100%; F1: zygote, 85%, and blastocyst, 78%) or the litter size in the P or F1 generation progeny (Fig. 2). Thus, culture of embryos from the zygote stage had no effect on the gross reproductive performance of progeny across several generations compared to blastocysts that were transferred without culture.
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The weights of the progeny were monitored in each generation (Fig. 3). There was no effect of progeny sex on growth to weaning (P > 0.05), so the weights of both sexes were considered together. Although there was no effect of treatment group on birth weight, there was an effect of treatment group on weight up to the time of weaning. In the P and F1 generations, the progeny that resulted from cultured blastocysts were lighter in weight than the in vivo blastocyst controls to the time of weaning. In the F1 generation, this difference was apparent only until Day 7. There was a significant difference in growth rate between the two treatment groups in the P (P < 0.001) and F1 (P < 0.001) generations, but not in the F2 generation (P > 0.05). There was a significant difference (P < 0.01) and a significant interaction (P < 0.01) between the groups in the P and F1 generations. This was also the case for the comparison of the P and F2 generations (generation effect, P < 0.05; interaction effects, P < 0.01). However, no differences were observed between the F1 and F2 generations. Since litter size was a significant covariate (P < 0.001), it was incorporated into the statistical model. There was no (P > 0.05) interaction effect between litter size and weight gain in the P generation, but there were significant effects in the F1 (P < 0.05) and F2 (P < 0.05) generations. Furthermore, there was no effect of the litter of origin of progeny on growth rate in the P and F2 generations (P > 0.05), but there was an effect of litter of origin in the F1 generation (P < 0.001).
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At necroscopy, the males were significantly heavier than the females (P < 0.001), but there was no effect of treatment (P > 0.05) on body mass. Animals were slightly smaller in the F1 generation than in the P generation (P < 0.01), but there was no interaction effect (P > 0.05) between treatment and generation or between treatment group and sex of progeny (Fig. 4). The weights of somatic organs (brain, pituitary, thyroid, liver, spleen, adrenal, kidney, and reproductive organs) at the time of necroscopy were assessed in the P and F1 progeny (Fig. 5). A summary of the outcomes from the statistical analyses for the organs, in which treatment group had a significant effect, is shown in Table 1. Since there was a significant effect of sex on some organ weights, the results for each sex are shown separately. The ages of the animals at necroscopy, body weights, and litter sizes were entered into the statistical model as covariates for all the analyses. In the P generation, thyroid weight was higher for in vivo blastocysts (P < 0.001), and there was no significant interaction (P > 0.05) with the sex of the progeny (Fig. 5A). In the F1 generation, the brains (P < 0.001), pituitaries (P < 0.01), and kidneys (P < 0.01) were heavier for the cultured blastocysts, with no interaction effect with sex (P > 0.05) (Fig. 5B). There was a significant interaction effect between treatment group and generation for pituitary weight (P < 0.05), with the pituitary being heavier in the F1 generation.
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In the P generation, the testes (P < 0.05) and epididymal (P < 0.01) weights were higher in progeny from the cultured blastocyst group, whereas this was not observed in the F1 generation (P > 0.05). In the F1 generation, only the prostate weight (P < 0.05) differed, being significantly higher in the in vivo blastocyst group (Fig. 6). The brain and spleen weights were significant covariates for testis weight (P < 0.02 and P < 0.05, respectively) and epididymal weight (P < 0.05 and P < 0.05, respectively). There was no effect of treatment group on uterine, vaginal, or ovarian weight (Fig. 7). Uteri were heavier in the F1 generation (P < 0.05), but there was no interaction effect (P > 0.05) between treatment group and generation (Fig. 7). Pituitary and liver weights were significant covariates for uterine weight (P < 0.05), and liver weight was a significant covariate for ovarian weight (P < 0.05). Female reproductive tract weight varies with the stage of the estrous cycle. In the present study, we did not assess the stage of the estrous cycle at necroscopy, so the result may be confounded by differences in the cycle between groups at the time of necroscopy.
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Perturbations of development in the early embryo have been reported to have impacts during postnatal life [1, 22, 23]. The causes are yet to be defined but may involve long-term metabolic reprogramming [22] or changes in the epigenetic control of gene expression (possibly at imprinted loci) [10]. Culturing of mouse embryos results in a defined phenotype of slower development [24], altered patterns of gene expression [15, 25], reduced numbers of cells in resulting embryos (with more cells undergoing cell death) [26, 27], and lower rates of long-term survival upon transfer [28, 29]. There have been few systematic studies of the long-term consequences of such treatment on the resulting progeny. This type of study is constrained by the absence of standardized tools for assessing or investigating the potential impacts of perturbation of the preimplantation embryo on long-term development and health. The OECD/OCDE 416 guideline is a widely used tool for the evaluation of potential chemical hazards on human health. The present study shows that a modified form of the assay may have utility for assessing the long-term impacts of perturbing preimplantation embryo development.
Culture of zygotes is a procedure common to almost all forms of ART. Progeny generated by IVF now constitute a significant proportion of the infants born each year in many countries. These babies have a higher risk of being born small [2], and epidemiological studies in other settings suggest that low birth weight affects homeostasis later in life [30]. In animal models, culturing has been reported to exert a range of effects, including perturbed birth weight [23], and it seems that changes in medium composition or culture conditions influence these outcomes [23, 31]. Embryo culture may also act as a selection pressure that favors the inheritance of some deleterious alleles [29].
The OECD 416 guideline provides a broad toxicity screen for a range of chemicals used in the environment. It was not designed for testing the effects of perturbing preimplantation embryo development, but we were interested to see if it could be used for this purpose. The present study found that culture from the zygote stage had no effect on the gross fertility of progeny across three generations. Nevertheless, changes in preweaning body weight and the size of some organs in mature animals were detected. Some of these effects persisted into the subsequent generation. It cannot be inferred that the specific changes observed in this model would necessarily be manifested in clinical populations. Yet these observations can be interpreted as evidence of some loss of integrity of the developmental program following culture of mouse zygotes. The observations provide the opportunity for investigation of the underlying causes of these deviations from the expected growth patterns, and they also provide a baseline for comparison that may allow empirical modifications of the culture techniques with the aim of alleviating these consequences. It will be of interest to determine how other perturbations during the preimplantation period of pregnancy influence outcomes in this system—for example, changes in maternal diet [1] or exposure to xenobiotics.
The small changes in body weight prior to weaning observed in the present study were accompanied by marked changes in mass for some organs in mature animals. Given the central role of the thyroid in setting the metabolic rate and nutrient utilization, it is possible that the smaller thyroid size in zygote progeny results in changes in body composition and overall homeostasis. The effect of embryo culture on preweaning weight persisted, in part, into the F1 generation. If confirmed, this may provide important new information about the long-term consequences of ART. Interestingly, in the F1 generation, the differences in thyroid mass were ameliorated, whereas the differences in brain, pituitary, and kidney mass were manifested, which suggests that different underlying causes for the changes in growth pattern exist in each generation.
The observed transgenerational effects of zygote culture on growth rates and organ mass represent an important finding of the present study and require further verification and investigation. The observed changes may be a consequence of epigenetic modification induced by zygote culture. There may also be metabolic explanations; for instance, differences in the lactational capacities of the mothers may result in differences in the growth rates of their progeny [32]. Interpretation of these putative transgenerational effects are complicated by the progressive loss of heterozygosity at individual loci in each generation. Furthermore, changes in epistatic interactions between B6 and CB alleles at multiple loci may occur with each generation. While any such effects might be expected to be similar for both treatment groups, they potentially confound the interpretation of the results. This putative transgenerational effect is best taken at this time as a preliminary finding until all potential genetic confounding effects are controlled for in future studies. Further analyses of the best genetic strategies to test this phenomenon are required.
The present study shows that there was no effect of zygote culture on the fertility of progeny. This provides some reassurance, since this is an effect that has been widely canvassed as a potential consequence of ART [33]. However, the present study was performed with mice of normal fertility, and therefore does not exclude effects due to the etiology of the underlying fertility within clinical populations. The present study does show an effect of zygote culture on testes and epididymal masses; further studies are required to determine whether these factors affect fertility under other circumstances, for instance, with advancing age or during IVF.
The present study shows that use of a modified form of the OECD 416 guideline for toxicity testing provides information that may be useful in guiding the assessment of the consequences of perturbing the normal growth of the preimplantation embryo. As a technically simple procedure that is suitable for standardization, it may provide the basis for an industry-wide tool for assessing the effects on progeny of all of the major inputs to ART. The standardized nature of the procedure provides a basis for comparing of results across testing laboratories and between different treatments of embryos, and may facilitate more rational approaches to the consideration of the safety profile of ART. It may also serve as an appropriate tool for examining more broadly the range of stressors that perturb early embryonic development for their effects on the well-being of progeny and subsequent generations.
ACKNOWLEDGMENTS
We thank Kim Gilliam (William Cook Australia P/L), Tomas Stojanov, and Sally Catt (Sydney IVF P/L) for helpful discussions, and the staff of Gore Hill Research Laboratories for the care and management of the animal colonies.
FOOTNOTES
1Supported in part by William Cook Australia P/L. 
Correspondence: 2FAX: 61 2 9926 6343; e-mail: chriso{at}med.usyd.edu.au
Received: 16 October 2006.
First decision: 25 December 2006.
Accepted: 3 August 2007.
REFERENCES
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  H. D. Morgan, X. L. Jin, A. Li, E. Whitelaw, and C. O'Neill The Culture of Zygotes to the Blastocyst Stage Changes the Postnatal Expression of an Epigentically Labile Allele, Agouti Viable Yellow, in Mice Biol Reprod, October 1, 2008; 79(4): 618 - 623. [Abstract] [Full Text] [PDF]
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