Sequence Diversity Lost in Early Pregnancy

Germline mutations are heritable; they occur before the formation of a fertilized egg and are found in all cells. They can be detected through somatic tissue sampling, and de novo mutations (DNMs) are well-studied. The majority of known DNMs originate in paternal cells, but some include maternal contributions as well. Certain kinds of DNMs prevent a fertilized egg from developing to term, and these are much less well-characterized. Some also lack sequence variants in certain genes and some are never observed in a homozygous form; these are also not well studied. Recombination failure can cause aneuploidies (trisomies or monosomies), and an estimated half of pregnancy losses are explained by this phenomenon. Early pregnancy loss is understudied, and there are few therapeutic interventions. This study, the Copenhagen Pregnancy Loss (COPL) study, was designed to contribute to the understanding of pregnancy loss through trios of patients (mother, father, and fetus) with clinically diagnosed pregnancy loss, attempting to document sequence diversity and interplay between meiotic recombination and point mutations. This study included 664 cases of early pregnancy loss with 1439 fetal samples (multiple were collected from each loss, where possible). In 467 of the 664 cases, there was at least 1 fetal sample and 1 sample from both parents. A total of 59 losses indicated a higher-than-expected kinship with the mother, and 11 indicated a higher kinship with the father. Whole-genome sequencing (WGS) was used to assess aneuploidies, and detected them in 206 cases. Of these, monosomy X and trisomy 16 were the most common. In addition, 19 large de novo copy number variants (CNVs) were detected in 14 loss cases, none of which were near a common fragile site. Of these 14 cases, 11 were euploid losses and 6 contained aneuploidies. Failure at meiosis I results in the presence of both homologous chromosomes from the same parent. An estimated 27.2% of paternal and 32.3% of maternal triploidies occur at recombination hotspots, supporting the idea of meiosis failure. A total of 15,086 DNMs were pinpointed as paternal and 5967 as maternal, consistent with previous literature supporting a high paternal contribution to DNMs. Consistent with this, paternal triploidies showed a proportionally higher paternal fraction of phased mutations. DNMs shown in maternal triploidies indicated a lower paternal fraction than euploid fetuses. In addition, there was no correlation between sister/homologous state differences for high-AB DNMs in paternal triploidies. When searching for pathogenic single-site variants (SSVs) in the DNMs, 26 genotypes were found that were pathogenic or likely pathogenic; a total of 23 were DNMs and 3 were biallelic predicted loss-of-function variants (pLoF). The frequency of pathogenic SSVs in early pregnancy loss was higher compared with controls [odds ratio (OR) 2.98, P=5.7×10-6), and this effect remained after correction for parental age. These results showed probable genetic causes for pregnancy loss in 254 of 467 cases, including aneuploidies, triploidies, pathogenic SSVs, and de novo CNVs. Most of the genetic causes of loss originated on maternal chromosomes, and fetuses with triploidies had significantly more DNMs than fetuses that were euploid. These results indicate significant sequence diversity in early pregnancy loss, with additional diversity likely present but unidentified in the stages between implantation and clinically recognized pregnancy. Future research should focus on potential explanations for early pregnancy loss that cannot be explained by genetic causes, as well as on potential interventions for these cases.