Aneuploidy Screening for Embryo Selection

Elpida Fragouli, Ph.D.; Dagan Wells, Ph.D.


Semin Reprod Med. 2012;30(4):289-301. 

In This Article

Chromosome Screening At the Blastocyst Stage of Preimplantation Development

The blastocyst stage is the final stage of embryonic development before implantation. Embryos become blastocysts ~5 to 6 days after fertilization, and 2 to 3 days after embryonic genome activation. Blastocyst embryos have undergone the first cellular differentiation and consist of an external layer of cells known as the trophectoderm (TE), a fluid-filled cavity known as the blastocoel, and an internal grouping of cells called the inner cell mass (ICM). After implantation, the TE will form the extraembryonic tissues (placenta, etc.), whereas the ICM will go on to form the fetus.

The culture of human IVF embryos until they reach the blastocyst stage is a relatively new innovation that has only become routine in recent years. Advances in extended embryo culture have used a combination of sequential stage-specific media with ultra-stable low oxygen culture systems.[75,76] Because embryos that become blastocysts in vitro have successfully overcome a series of potential challenges, they are considered to be of better quality and generally have an excellent implantation potential. For this reason, culture to the blastocyst stage is increasingly favored by IVF clinics worldwide. What is becoming increasingly clear, however, is that culturing embryos until the final stage of preimplantation development does not eliminate aneuploidy.

As with cleavage-stage embryos and oocytes, until recently FISH was the method of choice for the cytogenetic examination of human blastocysts in a research context.[77–81] Data obtained during these studies have shown that a multitude of chromosome errors, along with mosaicism, persist to the blastocyst stage, but their rates tend to be lower compared with the cleavage stage of preimplantation development.[77,81,82] It has been postulated that PGS at the blastocyst stage, via TE biopsy, may lead to improved clinical outcomes for infertile couples because better quality embryos would be screened.[2,52,83] Similar to PB biopsy, TE sampling is considered less damaging to the embryo than cleavage-stage analysis. Although it is typical to remove about five cells, the relative proportion of the embryo volume that is taken is smaller than that associated with single-cell biopsy at the cleavage stage. Furthermore, the clump of cells removed is destined to form the placenta rather than the actual fetus.

Jansen and colleagues[83] performed an RCT using five-color FISH to examine chromosomes 13, 18, 21, X, and Y of TE samples biopsied from blastocysts generated by younger (average maternal age: 33.5 years) infertile couples (test patient group A). Clinical outcomes were compared with a control patient group of similar characteristics (group B) but also with couples who were withdrawn from the RCT due to poor response (group C) and with couples who were eligible but did not want to participate in the RCT (group D). In the case of the principal control group (group B), blastocyst embryos had their zona breached, but no biopsy took place. Test and control groups B and D had agreed to undergo a single embryo transfer. Assessment of the results obtained did not show any significant improvement in the group that received PGS. The pregnancy rate seen for the test group A was 45.5%, whereas that seen for the principal control group B was 60.9%. For this reason the RCT was suspended and eventually terminated early. The authors postulated that the use of FISH to examine TE samples could have caused confusion and led to an overinterpretation and rejection of a significant number of good quality blastocysts.[83]

It was therefore evident that a different and more robust cytogenetic method was needed to clarify whether chromosome screening at the blastocyst stage could aid infertile couples to achieve a healthy pregnancy. Four different investigations reported results on the optimization and validation of CGH (metaphase or array) or SNP microarrays for the analysis of TE samples.[16,52,84,85]

The first two studies were performed by our group and involved the single or double TE biopsy of a total of 64 spare nontransferred blastocysts, followed by CGH and/or aCGH analysis. The remainder of the embryos were spread onto microscope slides and analyzed via FISH for chromosomes 13, 15, 16, 17, 18, 21, 22, X, Y, and any other that the CGH/aCGH had identified as abnormal. Another 10 blastocysts were disaggregated, and parts of their TE and ICM were placed into two different tubes. These were both analyzed by CGH.[16,52] Combination of these cytogenetic methods showed that 42% of analyzed blastocysts were uniformly normal in every cell, 30% were uniformly aneuploid due to one or more meiotic chromosome errors, 15.4% contained a mixture of different aneuploid cell lines (mosaic aneuploid), and 17% contained both normal and aneuploid cells (mosaic diploid aneuploid). Mosaic diploid-aneuploid blastocysts with >30% normal cells accounted for <6% of analyzed embryos. Additionally, 100% concordance was seen for the TE and ICM pairs.[16,52]

The other two studies[84,85] used SNP microarrays to examine the chromosome complement of a total of 102 nontransferred blastocysts that were also disaggregated into separate TE and ICM parts. The findings reported by Northrop and colleagues[84] were very similar to ours. Specifically, varying degrees of mosaicism were seen in 24% of the 51 investigated embryos, with the remaining 76% either uniformly euploid or aneuploid.[84] Moreover, good karyotypic concordance was observed between TE and corresponding ICMs.[84,85] It is therefore evident, that ~30% of blastocysts are mosaic, but only a relatively small proportion of these mosaics have as a majority a diploid cell line. Additionally, in most cases TE biopsies are accurate representations of the genetic constitution of the remaining of the blastocyst.[52,84,85] However, the main advantage these comprehensive molecular cytogenetic approaches have over FISH is that they are less susceptible to errors caused by mosaicism. Specifically, methods such as aCGH or SNP microarrays provide an average view of the biopsied TE sample, and aneuploidy is generally not detected unless it is present in more than a third of the cells sampled. It could be argued that the failure to detect low levels of abnormal cells is a disadvantage of these methods applied to blastocyst biopsies. However, in most cases, low-level mosaicism is probably of little clinical significance.

Because our validation work clearly demonstrated the reliability of CGH (metaphase or array) for TE analysis, we have been using this methodological approach in clinical cycles for the purpose of PGS and have accumulated data on 1300 embryos generated by >150 patients (average maternal age: 38.86 years; range: 29 to 47 years) (Fragouli[12]and unpublished). Abnormalities were scored in 58% of these blastocysts, and chromosomes from all groups were affected by aneuploidy, but the smaller ones (22, 16, 15, 21, and X) generally participated more frequently in errors. This observation is of potential interest because blastocyst chromosome anomalies seem to be similar to those scored using comprehensive methods for first and second PB analysis but are relatively different from findings reported after aCGH analysis of blastomeres from cleavage-stage embryos. Hence it is possible that most chromosome abnormalities that persist to the blastocyst stage originate in the oocyte and that embryos carrying large numbers of postzygotic errors (chaotic) arrest in culture before they reach this stage.

Improvements in clinical outcomes after the use of comprehensive methodology to examine the chromosomes of TE biopsies were seen during two different investigations. In the first study, performed by our group,[4] clinical outcome data obtained from 45 infertile couples who underwent blastocyst biopsy, followed by vitrification, CGH analysis, and transfer of euploid embryos in a subsequent cycle, were compared with outcomes from 113 couples receiving blastocyst transfer in the same IVF clinic during the same time period. Results obtained from this comparison showed that 68.9% of blastocysts transferred after CGH testing led to clinical pregnancies, whereas the figure was 44.8% for the cycles without aneuploidy screening. It was therefore concluded that the probability of an embryo selected for transfer producing a child is increased 1.5-fold.[4] An RCT is currently being planned to confirm these preliminary observations.

The second study was an actual RCT that used comprehensive aneuploidy screening of 24 chromosomes with the use of quantitative PCR (qPCR).[86] A total of 28 patients whose characteristics were <43 years of age and with one or more prior failed cycles participated in the trial. Thirteen of these patients (average maternal age: 34 ± 3.2 years) generated blastocysts that underwent chromosome analysis via qPCR (test group), whereas the remaining 15 (average maternal age: 32 ± 6.0 years) comprised the control group and did not receive the test. Fresh embryo transfers took place for both the test and the control groups. Clinical outcome comparisons between the two groups showed that the pregnancy rates were significantly higher in the test group (92%) than those seen in the control group (60%). Implantation rates were also improved in the test group compared with the control (75% versus 56%, respectively), but the difference was not statistically significant. The authors stated that this was an ongoing study.[86] [Table 4 and Table 5] summarize some of the studies discussed in this section.

Chromosome screening during the final stage of preimplantation development has given very promising preliminary results, but data from large RCTs (preferably multicenter) will be necessary to substantiate these observations.


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