P–427 Maternal and fetal thrombophilia mutations and the ANXA5 M2 haplotype are more prevalent in first trimester miscarriages of euploid embryos
Authors: Madjunkov M , Wilkinson M , Chen S , Abramov R , Trivodaliev K , Madjunkova S , Librach C 
Published: August 2021

Study question

Is there a cumulative effect of shared maternal and fetal thrombophilia mutations and the ANXA M2 haplotype in first trimester miscarriages and are there shared genotypes that may predict outcome?

Summary answer

Coexistence of MTHFR–677CT and PAI–1–4G/5G-mutations in euploid-fetus-mother -pairs may adversely affect early development.Maternal FII–20210GA and PAI–14G mutation were more prevalent in women who miscarried euploid-fetuses.

What is known already

Spontaneous abortion (SA) is a significant clinical problem with several different etiologies. Certain thrombophilia and folate-related gene mutations have been associated with an increased risk of miscarriage which may be due to impaired coagulation homeostasis. The M2-haplotype of ANXA5-gene promotor encoding the anticoagulation protein Annexin-A5 has also been implicated in early and recurrent SA. However, one important deficiency in the majority of the studies on this subject is a lack of information on the fetal/embryonic contribution to the thrombotic events during placentation and early embryo development. The purpose of this study was to evaluate the prevalence of specific and cumulative thrombophilia mutations in women experiencing first trimester (T1) miscarriages and in their fetal products of conception.

Study design, size, duration

This is a single Centre retrospective cohort study where we analyzed the frequency and cumulative effect of mutations in 10 thrombophilia/folate related genes and the ANXA5 M2 haplotype in first trimester miscarriages from 229 mother-product of conception (POC) pairs from Jan. 2018-Nov. 2020.

Participants/materials, setting, methods

All 229 POC DNA samples were confirmed to be fetal and had aneuploidy screening with NGS. We analyzed 10 different mutations in thrombophilia- and folate-related genes (Factor V-Leiden G1691A, Factor V-H1299R, Factor II-G20210A, Factor XIII-V34L, PAI-I–675 4G/5G, FGB–455G/A, MTHFR-C677T and -A1298C, MTR-A2756G, and MTRR- A66G) using single base sequencing methodology and the M2 haplotype of ANXA5 promoter by Sanger sequencing. Maternal–euploid fetus pairs were compared to maternal-aneuploid fetus pairs. SSPS software was used for statistical analyses, and p &lt; 0.05 with CI 95% was considered significant.

Main results and the role of chance

Aneuploidy was detected in 40.6% (93/229) of POC, and the rest were euploid. Women with euploid fetuses had a significantly higher FII20210GA mutation frequency than those who had aneuploid fetuses (p = 0.02). However, this difference was not detected in the fetuses. Minor allele frequency and genotype frequencies of MTHFR A1298C, MTR A2756G and MTRR A66G mutations as well as for Factor V-Leiden G1691A, Factor V-H1299R and mutations were similar between the groups studied. The frequency of the M2 ANXA5 haplotype was similar between the two groups (3.33% maternal-euploid fetus-pairs vs 3.1% in maternal-aneuploid fetus pairs). PAI-I –675 4G/4G genotype was more frequent in women with euploid fetal losses (25%) vs aneuploid (15%) (p = 0.05), but with no-significant difference in euploid (20%) vs aneuploid fetuses (13%). A cumulative effect of maternal and fetal mutations in MTHFR 677CT and PAI–14G was observed in maternal-euploid fetus pairs (p = 0.0011, OR 3.8 95%CI [1.8–11.3], and p = 0.05, OR 1.2 95%CI [1.12–3.6], respectively). Overall mutation load in tested samples was 4.14 mutations±1.5 [ 1.46±0.8-thrombophilia and 2.45±1.16-folate metabolism] and was similar between both groups of fetuses and their mothers.

Limitations, reasons for caution

The retrospective nature and cohort design limited the generalizability of our results. The comparison group is maternal-aneuploid fetus- pairs as the reason for the miscarriage is the chromosomal abnormality in the fetus. The ethnic background may be a cofounding factor that needs to be evaluated in a larger study which is ongoing in our center.

Wider implications of the findings: Our study found a high mutational load in the 10 folate metabolism and thrombophilia genes we studied in the maternal-fetus pairs. Our findings provide evidence that fetal contribution to placental thrombophilia should be considered in cumulative effect estimations of specific maternal mutations in improvement of management of early miscarriages.

Trial registration number

Not applicable