# Maternal Mosaicism Challenges in Non‐Invasive Prenatal Diagnosis

## TL;DR

This study shows that maternal mosaicism can interfere with non-invasive prenatal testing, affecting test accuracy and requiring careful evaluation.

## Contribution

The study highlights maternal mosaicism as a previously underappreciated challenge in NIPD for monogenic disorders.

## Key findings

- Maternal mosaicism was detected in 5.7% of NIPD_MD tests, preventing test use in those cases.
- Low-level mosaicism was confirmed through retrospective maternal sequencing analysis.
- Maternal mosaicism increases recurrence risk and requires alternative testing or counseling.

## Abstract

To report the incidental detection of maternal somatic mosaicism during the development of exclusion‐based non‐invasive prenatal diagnosis for monogenic disorders (NIPD‐MD) initially indicated for apparently de novo pathogenic or likely pathogenic variants.

A droplet digital PCR (ddPCR)‐based exclusion NIPD_MD assay was developed for four couples, each with a prior pregnancy affected by a rare autosomal dominant or X‐linked condition due to a de novo pathogenic or likely pathogenic variant. Assays were designed to detect fetal‐specific variants in maternal plasma, with validation performed on parental and proband samples.

In four cases, maternal somatic mosaicism (3%–9%) among 70 personalized NIPD_MD (5.7%) was identified during assay validation, rendering NIPD_MD infeasible due to interference from maternal alleles. Each couple was informed of the elevated recurrence risk. Depending on their preferences, invasive prenatal testing or intensive ultrasound follow‐up was undertaken. Retrospective analysis of maternal sequencing data confirmed low‐level mosaicism that had been filtered out during routine analysis.

These cases underscore a key limitation of exclusion NIPD_MD when maternal mosaicism is present. Its identification is essential for accurate recurrence risk estimation and genetic counseling. Sensitive detection methods, careful pre‐test evaluation, and transparent communication are critical to ensure informed reproductive decision‐making.

What is already known about this topic?◦Non‐invasive prenatal diagnosis (NIPD) using digital droplet PCR (ddPCR) on maternal blood is a popular alternative to invasive investigations. However, this test excludes alterations inherited from the mother.What does this study add?◦We report four cases of maternal mosaicism identified during the validation of a test in an NIPD context. Maternal mosaicism represents 5.7% of our tests to date, increasing the risk of recurrence and ruling out the use of a ddPCR test for NIPD.

What is already known about this topic?

Non‐invasive prenatal diagnosis (NIPD) using digital droplet PCR (ddPCR) on maternal blood is a popular alternative to invasive investigations. However, this test excludes alterations inherited from the mother.

What does this study add?

We report four cases of maternal mosaicism identified during the validation of a test in an NIPD context. Maternal mosaicism represents 5.7% of our tests to date, increasing the risk of recurrence and ruling out the use of a ddPCR test for NIPD.