Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

Abstract

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4-8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility.

Fig 1. Distributions of maternal and paternal ages and correlation between parents’ ages.

Colors indicate whether the maternal age refers to an egg donor or a non-donor patient.

Fig 2. Number of embryo biopsies submitted for PGS declines with maternal age for both day-3 blastomere biopsies and day-5 TE biopsies.

Error bars indicate standard errors of the means.

Fig 3. Rate of whole-chromosome abnormalities versus maternal age compared to fitted values from a logistic regression model.

Errors bars for the data indicate standard errors of the proportions, while error bars for the model indicate standard errors of the fitted means.

Fig 4. Schematic explaining the BPH signature of meiotic-origin chromosome gain.

Certain chromosomal signatures are indicative of meiotic versus mitotic origin of aneuploidy formation. The presence of three unmatched haplotypes (‘both parental homologs’; BPH) in any chromosomal region of the embryo suggests an error in either meiosis I (MI) or meiosis II (MII). Chromosome gains involving identical homologs (‘single parental homolog’; SPH) can arise either by mitotic error or MII errors in the absence of recombination [6, 36]. Reprinted from [36] with permission from Elsevier.

Fig 5. Heat maps depicted proportions of biopsies with different configurations of maternal and paternal chromosomes.

Minor aneuploidies and triploidies disproportionately involved maternal homologs. The paternal genome is absent from the majority of haploid embryos. Sub-diploid complements with missing maternal and paternal homologs were common in day-3 blastomere biopsies, but strongly depleted in day-5 TE biopsies. This results in a ‘steeper’ heat map surface for day-5 biopsies, centered on the euploid complement.

Fig 6. Venn diagram demonstrating that multiple forms of aneuploidy commonly co-occur within individual day-3 blastomeres.

Numbers represent percent of the total sample. Complex aneuploidies, especially those involving multiple forms of chromosome loss (highlighted in red) are common at day 3, but rare at day 5 of development. Isolated errors, in contrast, are relatively viable through blastocyst formation.

Fig 7. Maternal BPH error increases with maternal age.

Proportion of biopsies containing at least one maternal BPH error, stratified by sample type. Maternal BPH error increases with maternal age for both day-3 blastomere biopsies (β = 0.110, SE = 0.00404, P < 1 × 10⁻¹⁰) and day-5 TE biopsies (β = 0.120, SE = 0.00599, P < 1 × 10⁻¹⁰). Error bars indicate standard errors of proportions.

Fig 8. Rates of maternal BPH error are elevated on specific chromosomes.

A: Chromosomes 16, 22, 21, and 15 displayed elevated rates of maternal BPH error at both day 3 and day 5. B: Chromosome-specific rates of maternal BPH error are highly correlated at day 3 versus day 5 (r = 0.978, P < 1 × 10⁻¹⁰). Error bars indicate standard errors of proportions. C: Histogram of total affected chromosomes for biopsies with at least one maternal BPH error, but no putative mitotic errors. Colors indicate sample type.

Fig 9. Chromosome-specific rates of maternal BPH aneuploidy are negatively correlated with chromosome length.

Negative correlation was observed for both day-3 blastomeres (r = −0.623, P = 0.00148) and day-5 TE biopsies (r = −0.556, P = 0.00586). Error bars indicate standard errors of proportions.

Fig 10. Association between paternal BPH error and paternal age.

Rate of rare paternal BPH error was not associated with paternal age in day-3 blastomere biopsies (β = −0.00360, SE = 0.00933, P = 0.700), but displayed a weak negative association in day-5 TE biopsies (β = 0.0342, SE = 0.0146, P = 0.0194). Error bars indicate standard errors of proportions.

Fig 11. No significant association between putative mitotic error and maternal age.

No significant association was detected between rate of putative mitotic error and maternal age for either day-3 blastomere biopsies (β = −0.00186, SE = 0.00322, P = 0.564) or day-5 TE biopsies (β = 0.0119, SE = 0.00616, P = 0.0526). Error bars indicate standard errors of proportions.

Fig 12. No significant association between putative mitotic errors and paternal age.

No significant association was detected between rate of putative mitotic error and paternal age for either day-3 blastomere biopsies (β = 0.00315, SE = 0.00253, P = 0.213) or day-5 TE biopsies (β = 0.000540, SE = 0.00454, P = 0.905). Error bars indicate standard errors of proportions.

Fig 13. Chromosome-specific rates of aneuploidies of putative mitotic origin.

A: Chromosome-specific rates of putative mitotic-origin aneuploidy vary by chromosome. B: Rates of putative mitotic-origin aneuploidy were significantly correlated between days 3 and 5 (r = 0.762, P = 2.326 × 10⁻⁵). Error bars indicate standard errors of proportions. C: Histogram of total aneuploid chromosomes for biopsies affected with putative mitotic-origin aneuploidy, but no maternal BPH aneuploidies. Colors indicate sample type.

Fig 14. Chromosome-specific rates of putative mitotic errors are positively correlated with chromosome length in day-3 blastomeres.

Positive correlation was observed for both day-3 blastomeres (r = 0.734, P = 1.011 × 10⁻⁴) but not in day-5 TE biopsies (r = 0.351, P = 0.109). Error bars indicate standard errors of proportions.

Fig 15. Complex errors are more common in blastomere samples than TE samples.

A: Rate of non-euploidy according to total number of chromosomes affected, stratified by sample type. B: The relative difference between rates of non-euploidy affecting TE versus blastomere samples. More complex errors affecting greater numbers of chromosomes are increasingly rare among TE samples, suggesting inviability and/or self-correction of increasingly complex errors.

Fig 16. Associations between clinical indications for PGS and rates of meiotic and mitotic error detected with PGS, controlling for maternal age.

Only indications with at least one significant association are depicted, while full results are provided in S3–S8 tables. Effect size is measured by an odds ratio, where error incidence for a given referral reason is compared to error incidence for all other referral reasons. Error bars indicating 95% confidence intervals. Stars are used to indicate statistical significance in a logistic GLM: * P < 0.05, ** P < 0.01, *** P < 0.001. Translocation carriers had significantly higher rates of meiotic error than patients referred for other reasons. Patients with previous IVF failure had higher rates of mitotic, but not meiotic error, while patients with recurrent pregnancy loss had higher rates of meiotic (BPH) error at day 5.