Human Reproduction, Vol. 17, No. 9, 2362-2367,
September 2002
© 2002 European Society of Human Reproduction and Embryology
Fertilization in vitro increases non-disjunction during early cleavage divisions in a mouse model system
Department of Genetics and the Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA
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Abstract |
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BACKGROUND: We have been studying an unusual mouse—the BALB/cWt (Wt) male—in which the Y chromosome is susceptible to high rates of mitotic non-disjunction, particularly at the first two cleavage divisions. As these are the same divisions that human embryos generated through assisted reproductive technology must complete in an artificial setting, analysis of the Wt Y chromosome allows us to examine the effect of fertilization and culture in vitro on mammalian chromosome segregation. METHODS: We performed standard mouse IVF, cultured embryos in 5% CO2 in air or in a lowered oxygen atmosphere, and used fluorescence in-situ hybridization to examine the sex chromosome constitutions of 2-, 4-, 8- and 16-cell stage Wt Y-bearing embryos. RESULTS: We observed a significant increase in mosaic sex chromosome aneuploidy at each embryonic stage in embryos cultured in 5% CO2 in air, but under lowered oxygen conditions mosaicism returned to control (in-vivo) levels. CONCLUSIONS: Our results demonstrate that slight alterations in in-vitro conditions may have a considerable impact on the genetic quality of assisted reproductive technology-derived embryos and suggest that the genetic quality of embryos should be a fundamental concern in the development of new culture systems for clinical use.
Key words: fertilization in-vitro/non-disjunction/preimplantation embryo/Y chromosome
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Assisted reproductive technology has become increasingly important in the treatment of human infertility. In the 1995 National Survey of Family Growth, ~1.2 million women in the USA reported having had an infertility-related medical appointment within the previous year. Furthermore, US fertility clinics reported that in 1998, nearly 20000 deliveries (and 28500 babies) resulted from assisted reproduction. However, despite the success of these treatments, questions remain regarding their potential effects on embryo development.
For example, at least two types of observations suggest that human embryos derived from in-vitro procedures may be at some increased risk of chromosome abnormalities. First, although the introduction of ICSI has revolutionized the treatment of male factor infertility, several groups have reported an increased incidence of aneuploidy associated with ICSI-initiated pregnancies. For example, in an analysis of 1082 prenatal diagnoses performed on ICSI-initiated pregnancies (Bonduelle et al., 1998
) there was a 4-fold increase in de-novo chromosomal aberrations following ICSI. Second, in cytogenetic analyses of spare blastocysts from preimplantation embryos derived from human IVF, at least two groups have demonstrated high levels of aneuploidy. One study (Munné et al., 1994) found that 17% of morphologically normal 3 day embryos, considered at risk for an X-linked disease, were chromosome mosaics, and another (Ruangvutilert et al., 2000) reported mosaicism in >80% of arrested embryos or blastocysts cultured for 5 days.
Moral, ethical and legal issues complicate assessing the genetic quality of assisted reproductive technology-derived human conceptions. Therefore, appropriate animal models provide an important tool for studying potential effects of IVF and embryo culture on the health and development of mammalian embryos. To this end, the laboratory mouse has been widely used to model various aspects of human IVF. Recently, we provided data suggesting that a particular mouse model—the BALB/cWt (Wt) male—may be especially useful in examining errors of mammalian chromosome segregation in the in-vitro setting (Bean et al., 2001).
Male Wt mice carry an unusual Y chromosome that is prone to errors in segregation during early embryonic development. The Y chromosome on this inbred strain missegregates at an extremely high rate during the earliest cleavage divisions, so that by the 16-cell stage ~50% of Y-bearing embryos are sex chromosome mosaics (Bean et al., 2001). Interestingly, the frequency of Y non-disjunction is highest during the first two cleavage divisions, suggesting that these divisions may be particularly prone to chromosome missegregation (Bean et al., 2001). If this is a generalized feature of mammalian embryos, it raises a dichotomy for human assisted reproduction: the development of culture methodology to support preimplantation embryo development was an essential step in the revolution leading to current assisted reproduction methodology. However, if the early cleavage divisions of the human embryo are also error-prone, the very culture systems that spurred the development of the field might—at least under certain circumstances—compromise the genetic quality of the human embryo.
The Wt Y chromosome provides a tool for testing the hypothesis that fertilization and culture in vitro can affect the genetic quality of the mammalian embryo. Accordingly, we performed standard mouse IVF, cultured embryos through the first few cleavage divisions and assessed sex chromosome mosaicism to determine whether fertilization in vitro alters the frequency of Y chromosome non-disjunction. Our results indicate a highly significant increase in Wt Y chromosome missegregation in embryos derived in vitro. In fact, by the 16-cell stage ~70% of Wt Y-bearing embryos were found to be sex chromosome mosaics following fertilization and embryo culture in vitro under standard conditions.
In a second set of studies, we asked whether varying culture conditions, such as low oxygen concentration, alters Wt Y chromosome segregation; therefore, we conducted a series of experiments using different culture conditions. The observed sex chromosome mosaicism was reduced to in-vivo levels when fertilization and embryo culture were performed under reduced oxygen tension, which confirms previous studies in the mouse suggesting that lowered oxygen tension is beneficial to embryo development in vitro (Quinn and Harlow, 1978; Pabon et al., 1989; Umaoka et al., 1992; Eppig and Wigglesworth, 1995). More generally, our results demonstrate that slight alterations in in-vitro conditions can have a dramatic impact on the genetic quality of assisted reproduction-derived embryos and suggest that the genetic quality of embryos should be a fundamental concern in the development of new culture systems.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Mice
Breeding stock of BALB/cWt (Wt) and BALB/cBy (By) inbred strains was obtained from The Jackson Laboratory, and mice were housed in Thoren ventilated rack caging in a pathogen-free facility.
IVF and embryo culture
Fertilization in vitro and embryo culture were performed according to standard procedures. Depending upon the experiment, all media were equilibrated and embryos were cultured in either 5% CO2 in air or in an atmosphere of 5% O2, 5% CO2 and 90% N2 (5:5:90). To obtain sperm for fertilization, adult Wt or By males were killed and the epididymis and vas deferens removed from each testis and placed in prewarmed human tubal fluid (hTF) medium (Specialty Media). Mature sperm were released from the cauda epididymis and vas deferens and allowed to `swim out' for 15 to 30 min at 37°C in hTF medium containing 0.4% bovine serum albumin (BSA; Sigma).
To obtain oocytes, 4- to 6-week-old Wt or By females underwent ovulation induction with i.p. injections of 2.5–5 IU pregnant mares' serum gonadotrophin (Sigma) followed 44–48 h later by 5 IU of hCG (Sigma). Females were killed 13–15 h after hCG injection, and ovulated oocytes were released from the ampulla of the oviducts and placed into equilibrated hTF containing 0.4% BSA.
For fertilization, sperm and oocytes were mixed and incubated for 4–6 h. Fertilized oocytes were transferred to clean 200 µl drops of hTF medium under mineral oil (Roberts Laboratories) and cultured overnight. The following morning, all 2-cell embryos were transferred to 200 µl drops of potassium-enriched synthetic oviductal medium medium under oil and cultured for 4–54 h to obtain 2-, 4-, 8- and 16-cell stage embryos.
FISH analysis of preimplantation embryos
Prior to fixation, embryos were swollen gently in 0.03 mol/l citric acid (Sigma), transferred individually to microdrops of water on clean microscope slides, and fixed with drops of 5:2 methanol:glacial acetic acid. Slides were stored at room temperature for up to 1 week before processing for fluorescence in-situ hybridization (FISH) analysis.
A two colour FISH analysis was performed as described previously (Bean et al., 2001) using the Y-specific probe, pErs5 (Epplen et al., 1982) and the X-specific probe, DXWas70 (Disteche et al., 1985). Hybridized slides were detected with FITC–avidin and Rhodamine-labelled anti-digoxigenin to visualize the Y and X chromosome signals and were counterstained with DAPI (Sigma).
Slides were scored by two independent and blinded observers as described previously (Bean et al., 2001). For each embryo, the numbers of X and Y signals were scored in all metaphase and interphase cells. Mosaicism was defined as the presence of two or more different cell types in the same embryo; therefore, the presence of a single cell with a missing or additional Y chromosome was considered sufficient evidence for mosaicism.
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Results |
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Fertilization in vitro increases the frequency of Wt Y non-disjunction during early cleavage divisions
We previously reported data (Bean et al., 2001
) on the incidence of Y chromosome non-disjunction for 332 Wt Y-bearing preimplantation embryos derived from natural (in-vivo) matings (Table IA). To determine whether fertilization and culture in vitro increases the incidence of Y chromosome missegregation, we performed mouse IVF using standard conditions and examined aneuploidy levels in 2-, 4-, 8- and 16-cell preimplantation embryos. In total, we examined the sex chromosome constitutions of 149 Wt Y-bearing embryos (Table IB).
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Our analyses demonstrated a striking increase in aneuploidy among in-vitro-derived embryos by comparison with those developing in vivo (Figure 1
; Table IA,B
). The differences were significant for three of the four time-points examined (for the 4-cell stage, 
2 = 5.75, P < 0.05; for the 8-cell stage, 2 = 9.37, P < 0.01; and for the 16-cell stage, 2 = 4.40, P < 0.05). For the 2-cell stage, the difference did not reach statistical significance; however, we observed a 2-fold increase in aneuploidy among in-vitro-derived embryos, and it seems likely that, with additional data, the difference at the 2-cell stage would also reach significance. Therefore, we conclude that non-disjunction is increased in in-vitro-derived Wt Y-bearing embryos during early cleavage divisions.
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Wt Y chromosome non-disjunction is maximized during the first two cleavage divisions both in vivo and in vitro
The difference in aneuploidy rates between in-vivo and in-vitro-derived embryos could originate in one of two ways. First, Wt Y non-disjunction could be increased at each of the first four divisions and, possibly, at subsequent divisions as well; i.e. the in-vitro setting could impart an increased risk of non-disjunction regardless of the embryonic stage of development. Alternatively, the increase could be restricted to one or more of the earliest cleavage divisions, with in-vivo- and in-vitro-derived embryos experiencing similar rates of non-disjunction at later cell divisions.
To distinguish between these possibilities, we compared the cell-specific probabilities of non-disjunction—i.e. the likelihood that an individual cell in a 1-, 2-, 4- or 8-cell embryo will non-disjoin—for in-vivo- and in-vitro-derived embryos. We calculated the probability that an individual cell malsegregates at a given cell division, d (e.g. 2
4 cell), as Pd = [(
d+1 – d)/(1 – d)]/N, where d is the observed incidence of sex chromosome aneuploidy among all embryos at the initiating cell stage (e.g. the 2-cell stage) and N is the number of cells at the initiating cell stage. Using this approach to examine the data from in-vivo- and in-vitro-derived embryos, we estimated the cell-specific probability of Wt Y chromosome malsegregation at the first, second, third and fourth cell divisions (Figure 2).
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The results of this analysis demonstrate that, in both in-vivo and in-vitro settings, the liability to non-disjoin is effectively restricted to the first two divisions. Indeed, by the fourth cell division the rate of non-disjunction approaches 0, regardless of the method of fertilization or culture conditions. Furthermore, it is clear that the first two cell divisions are largely responsible for the difference in non-disjunction rates between in-vivo-derived embryos and those fertilized in vitro and cultured under standard conditions. That is, during both the first and second cleavage divisions the cell-specific rate of non-disjunction among the `standard' in-vitro-derived embryos was ~2 fold that of the in-vivo-derived embryos (i.e. 0.24 versus 0.12 at the first division, and 0.28 versus 0.16 at the second division); at the third division the rate of non-disjunction remained higher among the in-vitro- than the in-vivo-derived embryos (0.05 versus 0.03), but with both displaying low levels of malsegregation; and by the fourth division the rates of non-disjunction were negligible for both categories of embryo.
Lowered oxygen tension decreases Wt Y missegregation in vitro
We were interested in determining whether alterations in culture conditions affect Wt Y chromosome segregation. Since previous animal studies have demonstrated improved embryo quality under lowered oxygen tension (Quinn and Harlow, 1978; Pabon et al., 1989; Umaoka et al., 1992; Eppig and Wigglesworth, 1995), we decided to examine this variable. A second series of IVF experiments in which both fertilization and culture were conducted in an atmosphere of 5% O2, 5% CO2 and 90% N2 demonstrated a reduction in aneuploidy levels compared with embryos cultured using higher oxygen levels at each of the four cell stages examined (Figure 1; Table IB versus C). At two of these cell stages (the 4-cell stage, 2 = 8.96, P < 0.01; the 8-cell stage, 2 = 3.88, P < 0.05) the differences reached statistical significance. Moreover, there were no obvious differences between Wt embryos derived in vitro under reduced oxygen tension and those derived from natural matings (Table IA versus C). Thus, we conclude that alterations in culture conditions can affect the likelihood of Wt Y chromosome non-disjunction and—at least in this instance—can be used to reduce aneuploidy levels to those observed in vivo.
Significantly elevated Y chromosome non-disjunction is not a generalized feature of fertilization in vitro
In a final set of studies, we were interested in asking whether the in-vitro-associated liability to non-disjoin extends to `normal' Y chromosomes, i.e. Y chromosomes not known to be non-disjunction-prone. In previous studies we analysed sex chromosome aneuploidy levels in preimplantation embryos of By mice, an inbred strain related to Wt mice, and found no evidence for an increase in aneuploidy. Thus, sperm from By males were used to generate 74 Y-bearing in-vitro-derived embryos (Table IIB) for comparison with 130 previously reported By Y-bearing embryos derived from natural matings (Table IIA). Additionally, 92 By Y-bearing embryos were derived by fertilization and culture in vitro under reduced oxygen tension and compared with the two other types of By embryos. By Y-bearing embryos did not exhibit a significantly elevated incidence of sex chromosome mosaicism following fertilization and culture in vitro at either of the two oxygen tension levels (Figure 1; Table II). Therefore, we conclude that a predisposition for chromosome non-disjunction is necessary to observe the increase in mosaic aneuploidy produced in vitro; however, we cannot exclude the possibility that, given a larger sample size, we would have observed a slight but significant increase in aneuploidy in By Y-bearing embryos produced in vitro.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The non-disjunction-prone mouse Wt Y chromosome provides a unique tool for assessing the potential impact of fertilization and culture in vitro on the genetic quality of the mammalian embryo. The results of a relatively simple set of experiments using standard conditions for IVF suggest that subtle changes in the culture environment can significantly influence the fidelity of chromosome segregation during the early cleavage divisions.
These results have relevance to human assisted reproductive technology for two reasons. First, both our previous (Bean et al., 2001) and current studies of the Wt Y chromosome suggest that the early cleavage divisions of the mammalian embryo are particularly susceptible to errors of chromosome segregation. Since the vast majority of current assisted reproduction procedures necessitate in-vitro culture of the human embryo during this `vulnerable' period, our findings raise the possibility of iatrogenic error. Second, the demonstration that relatively minor changes in the culture environment can significantly increase the segregation error rate of a susceptible chromosome raises important questions about the remarkably high rates of mosaic aneuploidy reported in several previous studies of assisted reproductive technology-derived human embryos (Munné et al., 1994; Delhanty et al., 1997; Emiliani et al., 2000; Magli et al., 2000; Ruangvutilert et al., 2000).
It seems unlikely that assisted reproduction techniques are solely, or even primarily, responsible for this level of mosaicism. Indeed, our observations argue against this interpretation: in studies of mice with a `normal' Y chromosome (i.e. BALB/cBy males), we found no evidence that fertilization in vitro increased the risk of non-disjunction. Nevertheless, our observations indicate that, in situations in which there is a predisposing risk to non-disjunction, assisted reproduction may exacerbate the risk. In our study, the predisposition was imparted by an unusual non-disjunction-prone mouse Y chromosome. While no such chromosomes have been described in humans, we suggest that there are other factors that may impart a predisposing susceptibility. One such factor may be increased maternal age, long known to be the leading aetiological agent in human meiotic non-disjunction. In a molecular analysis of the origin of sex chromosome trisomies, a significant increase in maternal age in cases of post-zygotic origin (Thomas et al., 2001) was reported. The cases involved non-mosaic trisomies, suggesting that the non-disjunctional errors occurred in the first few cell divisions and implicating maternal age in the aetiology of very early mitotic, as well as meiotic, non-disjunction of the sex chromosomes. It is not yet clear whether this effect extends to autosomes since, other than trisomy 21, few trisomies of post-zygotic origin have been analysed molecularly. Furthermore, previous reports of mosaicism in human preimplantation embryos have provided little evidence linking age with embryonic mosaicism (Munné and Cohen, 1998). Nevertheless, it could be that the effect is restricted to specific types of non-disjunction (e.g. to premature separation of sister chromatids) or to specific chromosomes, and thus may have been missed in previous analyses. Additional, detailed analyses will be useful in determining whether maternal age increases specific categories of chromosomal mosaicism in assisted reproduction-derived preimplantation embryos, consistent with our predictions.
Our results further indicate that slight alterations in in-vitro conditions may have marked effects on the genetic quality of assisted reproduction-derived embryos. Specifically, we observed significantly reduced levels of mosaicism under lowered oxygen tension. Successful human assisted reproduction treatments are routinely performed at both atmospheric and reduced oxygen tensions. In human assisted reproduction, lowered oxygen has been suggested to improve preimplantation embryonic viability, although it has not been shown to increase pregnancy rates (Dumoulin et al., 1995, 1999). However, no data are available comparing the genetic quality of human preimplantation embryos under varying oxygen conditions. Therefore, it remains unknown whether altered oxygen conditions in human assisted reproduction procedures affect the incidence of aneuploidy in preimplantation embryos
Several studies have examined aneuploidy in IVF-derived bovine embryos (Kawarsky et al., 1996; Viuff et al., 1999; Slimane et al., 2000), but no data are available directly comparing aneuploidy levels at varying oxygen concentrations. Therefore, in this setting as well, it is not clear whether embryos produced and cultured at higher oxygen concentrations exhibit an increased incidence of aneuploidy.
Therefore, with the exception of the present report, a link between lowered oxygen tension and aneuploidy in preimplantation embryos has not yet been demonstrated. Nevertheless, we think it likely that the effect extends to mammalian species other than mice and that the failure to document it simply reflects the fact that chromosome constitution is seldom considered in assessments of assisted reproduction protocols. Furthermore, there is mounting evidence that variation in in-vitro culture conditions or in the maternal environment can have profound effects on the preimplantation embryo. For example, recent studies suggest that the composition of culture media can affect the expression and methylation status of imprinted genes in the preimplantation mouse embryo (Doherty et al., 2000; Khosla et al., 2001a,b; Ohno et al., 2001); that culture media can affect the ability of bovine embryos to progress to the late blastocyst stage, and do so in a gender-specific manner (Larson et al., 2001); and that maternal undernutrition limited to the preimplantation stage leads to variety of defects, including blastocyst abnormalities, in the rat (Kwong et al., 2000). We think it likely that these and other factors may influence chromosome segregation during the earliest cell divisions (Munné et al., 1997), either by acting on chromosome-associated proteins (e.g. centromere proteins) or on associated cellular structures (e.g. the spindle). Taken together with our observations, these studies underscore the complex requirements of the developing preimplantation embryo, and demonstrate the importance of examining all aspects of embryo health, including chromosome constitution, in future efforts to refine in-vitro culture systems.
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Acknowledgements |
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We gratefully acknowledge Dr Ron Conlon for providing an additional flow-meter and modular incubator chamber, Arlene Ilagan for technical assistance with the in-vitro fertilization procedure, and Elise Millie for assistance in scoring slides. This research was supported by research grants HD31866 (P.H.) and HD21341 (T.H.); C.B. was supported by training grant GM08056.
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Notes |
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1 To whom correspondence should be addressed at: Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA. E-mail: pah13{at}po.cwru.edu
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References |
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Submitted on February 12, 2002; accepted on May 9, 2002.
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