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

Department of Clinical Immunology, Odense University Hospital, DK, Department of Clinical Immunology at Aalborg University Hospital, Northern DK

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.

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.

sRPL patient: women. Sibling: Older brother to sRPL patient. Child: The sRPL patient's child(ren) of both sexes. Biological sex is used.

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