Microchimerism in Patients With Recurrent Pregnancy Losses

Microchimerism From a Firstborn Boy or an Older Brother in Patients With Recurrent Pregnancy Losses: a Pilot Study

This pilot study aims to evaluate if microchimeric cells in a patient with recurrent pregnancy loss (RPL) can be detected by the blood analysis for the presence of the DYS14 gene and the use of indel-panel methods and also, to examine if this method can distinguish the cell's origin; comparing gene sequence from the patient's firstborn son or her older brother. In addition, the pilot study will provide the investigators with information and experience necessary for a subsequent main study to be conducted.

Study Overview

• Status: Recruiting
• Conditions: Recurrent Pregnancy Loss, Not Pregnant
• Intervention / Treatment: Genetic: Blood sample; Genetic: Swap test

Detailed Description

To the investigators' knowledge, no previous study has determined the origin of microchimeric cells, and therefore, this pilot study will use a newly developed genetic analysis which will compare DNA fragments from the male microchimeric cells with DNA fragments from the RPL patient's son(s) and older brother(s).

The pilot study aims to evaluate the functions and capacity of a newly developed genetic test identifying microchimeric cells. A pilot study is necessary to assure the DNA fragments (indels) analyzed in the genetic analysis include enough informative differences to distinguish between the son(s), daughter(s) and the older brother(s) before these investigations can be initiated in a larger sample. To assure the test can distinguish origin of microchimeric cells between relatives and work independent of gender, The study will include both the proband's daughter(s) and son(s) to strengthen the confidence that the test possesses this ability.

10 sRPL and their older brother(s) and firstborn child is included. total included: approx 30.

Only sRPL whose brother(s) and child(ren) also consent to participate, will be included.

Blood sample of 12 ml EDTA plasma is collected from the sRPL patient. A swap sample from oral mucosa is collected from the older brother and child(ren).

The samples will be centrifuged and the buffy coat containing the DNA will be collected from patient and stored at -80 °C. To detect the multi-copy DYS14 marker located in the TSPY1 gene on the Y-chromosome a real-time PCR analysis will be performed on the extracted DNA with a PCR mastermix specific for the reference gene.

Also, The extracted DNA will be amplified using specific primers and their associated probes in a multiplex PCR analysis. The specific primers target the 10 different indels leading to 10 PCR products with non-overlapping amplicon sizes. After the PCR analyses, the fragments are analyzed by capillary electrophoresis using GeneticAnalyzer and GeneMapper. When differences are identified, a qPCR analysis is performed on samples from only the proband with primers and probes specific for the indel fragments that are only present in either the child or the older brother. 12 wells loaded with 30.000 GE in each well are screened along with a no-template control and a positive control containing 10 GE of DNA homozygous for the allele variant.

• Study Type: Interventional
• Enrollment (Anticipated): 30
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Caroline Noergaard-Pedersen, MD; Phone Number: +4541120267; Email: c.noergaardpedersen@rn.dk
• Study Contact Backup: Name: Ole Bjarne Christiansen, professor; Phone Number: 41120267; Email: olbc@rn.dk

Study Locations

• Denmark
  • Aalborg, Denmark, 9000
    • Recruiting
    • Aalborg University Hospital
    • Contact: Caroline Nørgaard-Pedersen; Phone Number: 41120267; Email: c.noergaardpedersen@rn.dk

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

Description

sRPL patient (proband):

Inclusion Criteria:

• ≥3 consecutive pregnancy losses ≤12 weeks of gestation, which do not include confirmed ectopic or molar pregnancies.
• Age <41 years at time of admission
• Patients who have minimum one alive older brother with a common mother and prior birth of a boy, and patients who have minimum one alive older brother with a common mother and prior birth of a girl.

Exclusion Criteria:

• Significant intrauterine malformations
• Thyroid dysfunction
• Known chromosomal abnormality
• An older brother, a son or a daughter from whom we cannot not collect a swab test e.g., due to lack of consent, death, distance etc.
• Transplant recipient
• Transfusion recipient
• Pregnancy at the time the blood sample is collected

Older Brother:

Inclusion Criteria:

• Age difference between proband and the older brother <15 years
• Common mother with the proband

sRPL patient's child:

Inclusion Criteria:

• Age <15 years

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: sRPL patient; sRPL with an older brother and at least one child prior to diagnosis	Genetic: Blood sample; Blood sample of 12 ml EDTA plasma.
Experimental: Brother or child to the sRPL patient; An older brother (with at least same biological mother) og child to the sRPL patient	Genetic: Swap test; receive swab tests for collecting cells from the oral mucosa

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Frequency of sRPL patients with microchimeric male cells	Prevalence of sRPL patients with microchimeric male cells in the peripheral blood	Non-pregnant state, performed during study completion, i.e. within 1 year
Frequency of sRPL patients with microchimeric cell originating from older brother	Determination of microchimeric cell origin: brother, son, daughter or not identified	Non-pregnant state, performed during study completion, i.e. within 1 year
Frequency of sRPL patients with microchimeric cell originating from the patient's son	Determination of microchimeric cell origin: brother, son, daughter or not identified	Non-pregnant state, performed during study completion, i.e. within 1 year
Frequency of sRPL patients with microchimeric cell originating from the patient's daughter	Determination of microchimeric cell origin: brother, son, daughter or not identified	Non-pregnant state, performed during study completion, i.e. within 1 year
Frequency of sRPL patients with microchimeric cell originating from unknown person	Determination of microchimeric cell origin: brother, son, daughter or not identified	Non-pregnant state, performed during study completion, i.e. within 1 year

Collaborators and Investigators

• Sponsor: Caroline Nørgaard-Pedersen
• Collaborators: Department of Clinical Immunology, Odense University Hospital, DK; Department of Clinical Immunology at Aalborg University Hospital, Northern DK
• Investigators: Principal Investigator: Caroline Nørgaard-Pedersen, MD, Aalborg University Hospital, Denmark

Study record dates

Study Major Dates

• Study Start (Actual): April 1, 2022
• Primary Completion (Anticipated): January 1, 2023
• Study Completion (Anticipated): January 1, 2023

Study Registration Dates

• First Submitted: April 8, 2022
• First Submitted That Met QC Criteria: April 15, 2022
• First Posted (Actual): April 22, 2022

Study Record Updates

• Last Update Posted (Actual): April 22, 2022
• Last Update Submitted That Met QC Criteria: April 15, 2022
• Last Verified: April 1, 2022

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Pathologic Processes; Disease Attributes; Pregnancy Complications; Death; Recurrence; Abortion, Spontaneous; Abortion, Habitual; Fetal Death
• Other Study ID Numbers: F2022-012

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No