Prenatal Analysis of Cell-free Circulating Fetal DNA (APAFLC)

September 15, 2025 updated by: University Hospital, Bordeaux

Development of a Protocol for Non-Invasive Prenatal Analysis of Cell-Free Circulating Fetal DNA (ccfDNA) Using Massively Parallel Sequencing (MPS)

In criminal cases involving sexual assault, victims may later discover they are pregnant, creating profound uncertainty about the paternity of the fetus. These distressing situations compound the trauma already experienced by the victims. To address this, the Medico-Legal Hematology Laboratory (LHML) in Bordeaux aims to validate a method based on Massively Parallel Sequencing (MPS) and a specific isolation protocol for analyzing cell-free circulating fetal DNA (ccfDNA) in maternal blood samples. This identification process will confirm or exclude paternal genetic relationships between the fetus and potential fathers between 6 and 22 weeks of amenorrhea. These analyses will only be conducted under judicial requisition, in accordance with the French Code of Criminal Procedure

Study Overview

Status

Recruiting

Conditions

Intervention / Treatment

Detailed Description

The objective of this study is to validate a non-invasive protocol for extracting and purifying ccfDNA from maternal blood samples and analyzing fetal DNA using MPS technology to establish paternity. To develop this methodology, a preliminary study will be conducted using blood samples from pregnant women whose partners' identities are known. LHML has partnered with Professor Sentilhes' team at Bordeaux University Hospital Center to obtain these samples.

Venous blood samples (~10 mL) will be collected from pregnant women between 6 and 22 weeks of amenorrhea in PAXgene Blood ccfDNA tubes (Qiagen) to prevent DNA degradation. The samples will be centrifuged to separate plasma, and ccfDNA will be isolated using the EZ2 Connect Fx instrument and the EZ1&2 ccfDNA kit (Qiagen). In parallel, buccal swabs will be collected from the mothers and their partners as reference samples, processed using LHML's routine DNA extraction protocols. Additionally, umbilical cord blood from newborns will be collected and processed with an in-house protocol to obtain purified DNA extracts.

These reference samples will allow us to confirm the profiles previously identified for the pregnant women and the fetuses but also to validate the filiation analysis between the fetus and his father. All DNA samples will be quantified using the Investigator QuantiPlex Pro kit (Qiagen) and the QuantStudio 5 Real-Time PCR System (Applied Biosystems).

ccfDNA from maternal blood will be analyzed using the MiSeq FGx Sequencing System (Verogen), the first NGS instrument validated for forensic genomics. The Verogen ForenSeq MainstAY Kit, which amplifies and sequences 27 autosomal STRs, 25 Y-STRs, and Amelogenin, will be used. This approach ensures reliable profiling even from low concentrated ccfDNA samples, with capacity for up to 96 libraries per run.

Reference DNA from buccal swabs and umbilical blood will undergo MPS using the same Verogen ForenSeq MainstAY Kit and be cross-validated with capillary electrophoresis using the Investigator 24Plex QS kit (Qiagen). Data analysis will be performed with the ForenSeq Universal Analysis Software and an in-house parentage analysis program developed by LHML. The Familias software may also be used to calculate likelihood ratios for paternity determination. All data processing systems are offline to ensure confidentiality.

This open prospective study will require samples from 20 complete triads (mother, father, newborn) and will address two major critical points. The first objective is to determine the quality threshold of the genetic profiles obtained with the Verogen ForenSeq MainstAY kit necessary for reliable paternity analysis. This will be assessed by calculating the percentage of validated STR loci among the 27 autosomal STRs analyzed. The second focus is to establish the optimal ccfDNA quantity required to generate a genetic profile covering at least 20 autosomal STRs using the Verogen ForenSeq MainstAY kit. This will involve quantifying the ccfDNA extracts and correlating these values with the maternal blood volume needed to obtain sufficient ccfDNA for analysis. Ultimately, this study aims to advance scientific understanding of ccfDNA analysis in pregnant women using MPS technology and, most importantly, to provide critical support to women who become pregnant following sexual assault by enabling them to confirm the paternity of their fetus with certainty, addressing a crucial aspect of their psychological recovery

Study Type

Interventional

Enrollment (Estimated)

60

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Locations

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

Yes

Description

Inclusion Criteria:

  • Age 18 or older.
  • For woman: pregnancy between 6 and 22 weeks
  • For both mother and father: non-opposition to participate and genetic signed informed consent

Exclusion Criteria:

  • Unknown paternity.
  • Maternal blood-borne infections (HIV, syphilis, hepatitis B).
  • Inability of the father to provide a sample.
  • Insufficient comprehension of French by either parent.
  • Mother or father under legal protection

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Other
  • Allocation: N/A
  • Interventional Model: Single Group Assignment
  • Masking: None (Open Label)

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
Experimental: Genetic profile
Filiation analysis between the fetus and his father
Other: Genetic profile
Filiation analysis between the fetus and his father

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Quality of Genetic profile
Time Frame: Baseline
The quality of the genetic profile obtained with the ForenSeq MainstAY kit will be estimated as a percentage of validated markers (loci) among the 27 autosomal markers analyzed by the kit
Baseline

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Free circulating fetal DNA
Time Frame: Baseline
The quantity of circulating cell-free fetal DNA necessary for the establishment of a genetic profile will be measured from a minimum of 20 autosomal markers using the ForenSeq MainstAY kit This value will be measured by the concentration in ng/µl.
Baseline
Maternal blood
Time Frame: Baseline
Quantity of maternal blood necessary to obtain a sufficient quantity of circulating cell-free fetal DNA for genetic analysis
Baseline

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

University Hospital, Bordeaux

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

September 15, 2025

Primary Completion (Estimated)

September 1, 2026

Study Completion (Estimated)

September 1, 2026

Study Registration Dates

First Submitted

March 3, 2025

First Submitted That Met QC Criteria

March 3, 2025

First Posted (Actual)

March 7, 2025

Study Record Updates

Last Update Posted (Estimated)

September 16, 2025

Last Update Submitted That Met QC Criteria

September 15, 2025

Last Verified

September 1, 2025

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • CHUBX 2024/80

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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