Introduction

Extracellular nucleic acids have already been known to exist in the blood circulation since 1948 . The observation of abnormally high concentrations of DNA in serum of cancer patients lead Stroun et al. to suggest that DNA found in the serum of cancer patients might be released by tumor cells . This hypothesis was confirmed when tumor-derived oncogene mutations were identified in the plasma and serum of patients with hematological malignancies . Since then, analysis of cell-free DNA in plasma identified copy number alterations, single nucleotide variants, and viruses associated with several types of cancers . Similarly, the placenta is considered by many a pseudomalignant tissue, invading the uterine epithelium to enable embryo implantation while releasing fragments of DNA in the maternal plasma as part of a physiological placental cell turnover during pregnancy . Inspired by this theory, Lo et al. discovered Y-chromosomal sequences in the plasma of pregnant women with male fetuses in 1997 . In healthy pregnant women, the majority of plasma DNA is a result of apoptosis or necrosis of maternal cells from multiple tissues . In contrast, “fetal” DNA constitutes only a minor fraction of approximately 5%–20% of cfDNA in plasma during the first trimester, and originates mainly from the cytotrophoblast cells of the placenta . Therefore any noninvasive prenatal test contains a major signal from the maternally derived DNA and a minor signal from the placenta-derived DNA. When applying genome-wide sequencing of plasma cfDNA not only the placental genome and potential aneuploidies in the conceptus are identified, but also the maternal genome is scanned for differences compared to the normal.

Early reports on the size of cfDNA using qPCR have shown that placenta-derived DNA fragments were generally shorter than the maternal ones . More recently, paired-end sequencing has allowed to determine the exact size of millions of cell-free DNA fragments in plasma and distinguished unique placental reads from the high background of maternal DNA. The predominant size of plasma DNA of pregnant women is 166 bp followed by a series of smaller peaks that present at a periodicity of 10 bp at sizes of 143 bp and shorter . It has been shown that the shorter placental DNA fragments are overrepresented in the fraction with fragments smaller than 150 bp . It has also been shown that DNA degradation is not random but occurs by enzymatic processes at specific sites between nucleosomes, the internucleosomal linker sites. These patterns of cfDNA release have been studied extensively by two groups. Snyder et al. demonstrated that nucleosome footprints in cfDNA are tissue specific. They inferred nucleosome spacing from cfDNA in healthy individuals and demonstrated that it correlates with epigenetic features of lymphoid and myeloid cells. This finding reinforces the idea that hematopoietic cell death is the main source of cfDNA . The group of Lo performed plasma DNA tissue mapping, a technique based on genome-wide bisulfite sequencing of plasma DNA. This analysis revealed the major tissue contributors of circulating DNA in both healthy nonpregnant and pregnant women as well as in patients with cancer. Similarly to Snyder et al. they found that > 70% of the circulating DNA is derived from white blood cells, neutrophils, and lymphocytes. The remaining fraction is mainly derived from the liver and, in pregnant women, the placenta. The authors speculate that with further refinements in the deconvolution algorithm, it might become possible to identify the contribution of other tissues to the DNA pool . Hence, localization of the cfDNA fragments can reveal the tissue of origin that contributes to cfDNA in both healthy and pathological conditions.

Maternal Constitutional Copy Number Variations

Because maternal DNA constitutes the major DNA fraction of cfDNA, it may not be surprising that discrepancies of the maternal genome with respect to the reference genome can interfere with the interpretation of cfDNA-based prenatal testing results. Maternal constitutional copy number variations (CNVs) have been a major contribution to false positive cfDNA-based prenatal test results . Our group was the first to show that CNVs as small as 500 kb can cause trisomy z -scores to pass the accepted cutoff of three leading to false positive or false negative results . The group of Snyder demonstrated that a trisomy 18 discordancy between noninvasive and invasive prenatal test results was caused by a constitutional large duplication on the maternal chromosome 18 and, in another case, by a smaller maternal CNV of approximately 500 kb . In a recent systematic review of the literature it was compiled that among the 60 false positive trisomy cases, 29 (48%) had an underlying maternal CNV . Zhou et al. showed that there is a strong correlation of higher z-scores with increasing size of maternal CNVs . Similarly, maternal X chromosomal CNVs have been reported to be the cause of false positive sex chromosome aneuploidy cfDNA-based prenatal test results .

Because placental DNA constitutes only a small fraction of plasma cfDNA, not only constitutional (i.e., present in all cells of the body), but also somatic CNVs (i.e., present in only a subset of all cells) can cause false positive and, theoretically, false negative results. Maternal (segmental) chromosomal mosaicism is another major cause of discordant results of cfDNA screening and even low-grade mosaicism in the maternal genome can skew the z -statistics significantly. Bayindir et al. identified a maternal mosaic segmental deletion on chromosome 13 by applying genome-wide analysis. This deletion was present in only 17% of the maternal blood cells analyzed by FISH . Although rare, similar large mosaic maternal duplications of the chromosome 18 long arm have been observed and they caused false positive cfDNA test results . In another case with a false positive cfDNA test for fetal trisomy 18, the mother was identified with a supernumerary ring chromosome 18 in 35% of her cells .

Cell-free DNA-based prenatal testing in pregnancies with sex chromosome aneuploidies (SCAs) has lower sensitivities and specificities as compared to testing for the common fetal trisomies . In a prospective study, Wang et al. have shown that maternal chromosome X mosaicism was the main cause of discordant sex chromosomal aneuploidy . In another study by the same group, cfDNA Noninvasive Prenatal Testing (NIPT) findings suggested a case with lower X chromosome concentration. Maternal karyotyping showed mosaicism for monosomy X (mosaic Turner syndrome), indicating that this maternal mosaicism masked the true contribution of fetal chromosome X to the cell-free DNA pool .

The relatively high frequency of maternal CNVs and maternal sex chromosome mosaicism warrants maternal white blood cell testing with array-CGH or FISH to avoid unnecessary invasive testing . Most maternal CNVs are benign or of unknown clinical significance .

Maternal Malignancy

Almost 20 years after the discovery of tumor-derived cell-free DNA in plasma of cancer patients these notable findings were also observed in the pregnant population. In 2013, false positive results with unexplained trisomy 13 and monosomy 18 lead scientists to look beyond the fetal genome and identified a metastatic small cell carcinoma postpartum . A unique pattern of segmental chromosomal imbalances identified in cfDNA-based NIPT and persistent in consecutive samples prompted Vandenberghe et al. to suspect a malignancy. Whole body MRI and subsequent molecular characterization identified a non-Hodgkin lymphoma as the underlying cause of the aberrant pattern . Amant et al. showed that plasma DNA profiling during cfDNA NIPT identifies approximately 1 cancer per 2000 women who were not yet aware of the malignancy. Molecular characterization of the CNVs confirmed the cfDNA variation to be tumor derived. . Sun et al. used plasma DNA tissue mapping and identified a case of follicular lymphoma based on the observation that the contribution of B-cells was unexpectedly higher in circulating plasma DNA . More case reports describing the detection of cancers during pregnancies are emerging. A woman with a positive cfDNA NIPT result for full or partial monosomies of chromosomes, 13, 18, 21, and X was subsequently found to have hepatic lesions and was postpartum diagnosed with late stage colon cancer . Similarly, a multiple myeloma was identified during routine cfDNA-NIPT showing subchromosomal imbalances involving seven autosomal chromosomes . In a larger retrospective study by Bianchi et al. seven out of the 10 cases with occult maternal malignancies had a cfDNA NIPT result with unique patterns of nonspecific copy-number gains and losses across multiple chromosomes . In an effort to determine the underlying biological cause in 37 abnormal cfDNA NIPT reports with multiple aneuploidies, follow-up analysis revealed a maternal malignancy in 19% of the cases .

Amant et al. estimate the incidence of cancers in pregnant woman, unsuspected to have a cancer, to be at least 1/2000. Hartwig et al. reviewed the literature and estimate that 15% of unexplained false positive cases were caused by maternal malignancies . Diagnosis of a malignant disease in pregnancy is often delayed because symptoms are attributed to pregnancy . Detection of malignancy and its management during pregnancy presents a conflict between optimal maternal therapy and fetal wellbeing . Recent data from a multicenter control study suggest that prenatal exposure to maternal cancer with or without treatment did not impair the health and general development of children . Therefore early diagnosis and appropriate management of pregnancy-associated cancer can accelerate treatment and improve both maternal and neonatal outcome .

Other Maternal Disorders

Because the cell-free DNA is derived from multiple tissues, diseases in other organs or organ systems that result in apoptosis and DNA shedding in the circulation may well skew prenatal testing results.

It has been demonstrated that upon solid organ transplantation, DNA from the transplant can be detected in the cfDNA . More importantly, it has been shown that this DNA fraction increases upon graft rejection . Not surprisingly, pregnant women with an organ transplant are at risk of shedding large amounts of graft DNA in the plasma. This, in turn, will reduce the overall “fetal” DNA fraction which could subsequently cause a misdiagnosis. Discordances in fetal sex determination between cfDNA prenatal testing and ultrasound imaging have been described in two separate reports so far . In both cases, the pregnant women had undergone a transplant from a male donor prior to pregnancy. cfDNA NIPT results reported a male pregnancy in contrast to fetal anatomy ultrasound showing female genitalia. Follow-up testing confirmed the fetuses to be female. Hence, apoptosis of cells derived from the transplanted organ lead to the detection of Y-chromosome sequences and, as a consequence, to incorrect calling of the fetal sex. This type of erroneous fetal sex assessment may be avoided using bioinformatics cfDNA analysis pipelines that can identify unusual fetal fractions or which compare different approaches of fetal fraction estimation .

Systemic lupus erythematosus (SLE) was one of the first diseases reported to be associated with elevated circulating plasma DNA concentrations . Paired-end massively parallel sequencing of plasma DNA of nonpregnant patients with active SLE showed aberrations in measured genomic representations (MGRs), this is significantly more or less DNA fragments aligned to the reference genome as compared to controls. In addition, the size distribution profiles of SLE patients show elevated proportions of shorter fragments (< 115 bp) and hypomethylation compared to healthy controls . The aberrations in MGR and shortening of plasma DNA fragments correlated with disease activity. The authors hypothesize that preferential binding of antidouble strand DNA antibodies present in SLE patients with active disease hinders clearance of these short fragments from the circulation. SLE is one of the most common autoimmune disorders in women during their childbearing years and therefore another possible cause of abnormal cell-free DNA genomic profiles.

Finally, abnormal genome-wide representation profiles have been reported in a pregnant woman with severe vitamin B12 deficiency. After treatment with parenteral vitamin B12, the abnormal genome-wide profiles normalized . Vitamin B12 deficiency is known to cause ineffective erythropoiesis and intramedullary hemolysis. Although this is a single case report, the authors speculated that the abnormal profile was a consequence of the vitamin B12 deficiency. In Table 1 we summarize the main causes of abnormal or unexpected findings identified by cell-free DNA prenatal screening caused by maternal genomic alterations.

Table 1

Overview of Maternal Acquired or Constitutional Genomic Alterations That Can Cause Discordant cfDNA-Based Prenatal Screening Results

Condition	References
Maternal CNV	Zhang et al. , Bayindir et al. , Snyder et al. , Brison et al. , Meschino et al. , Clark-Ganheart et al.
Maternal mosaicism	Ganheart , Bayindir et al. , Flowers et al. , Wang et al.
Maternal malignancy	Osborne et al. , Vandenberghe et al. , Bianchi et al. , Snyder et al. , Smith et al. , Imbert-Bouteille et al.
Nonmalignant maternal medical disorder	Schuring-Blom et al. Chan et al.
Transplantation	Neufeld-Kaiser et al. , Neofytou et al.

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