Maternal vs Fetal Origin of Placental Intervillous Thrombi

Abstract

Objectives: To determine maternal vs fetal origin for blood in placental intervillous thrombi (IVTs).

Methods: We used comparative analysis of microsatellites (short tandem repeats [STRs]), sex chromosome fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC) for fetal (ɑ-fetoprotein [AFP]) and maternal (immunoglobulin M [IgM]) serum proteins to distinguish the origin of IVTs. Using an informatics approach, we tested the association between IVTs and fetomaternal hemorrhage (FMH).

Results: In 9 of 10 cases, the preponderance of evidence showed that the thrombus was mostly or entirely maternal in origin. In 1 case, the thrombus was of mixed origins. STR testing was prone to contamination by entrapped fetal villi. FISH was useful but limited only to cases with male fetuses. IgM showed stronger staining than AFP in 9 cases, supporting maternal origin. By informatics, we found no association between IVTs and FMH.

Conclusions: Evidence supports a maternal origin for blood in IVTs. IHC for IgM and AFP may be clinically useful in determining maternal vs fetal contribution to IVTs.

Keywords: COVID-19; FISH; Immunohistochemistry; Perinatal; Placenta; STR; Thrombi.

Figure 1

Identity testing via short tandem repeat (STR) analysis using 2 illustrative color-coded loci. Although no maternal DNA was available for comparative analysis, the genotype of thrombus cells can be determined by sizing the peaks on thrombus vs fetal electropherograms. The fetus profile matches the thrombus at 1 allele for each locus (double-headed arrows) corresponding to the allele inherited from the mother (M) with the other allele being paternally inherited (P). One of thrombus alleles (labeled with *) is not present in the fetus profile; thus, it is not of fetal origin and must have been of maternal origin.

Figure 2

Comparative molecular genotyping of fetal tissues vs clot areas. Electropherograms of all tested short tandem repeat (STR) loci and the amelogenin sex typing are from the AmpFLSTR Identifiler PCR amplification test (ThermoFisher Scientific). The bottom panels show a set of peaks from fetal DNA. The top panels show a set of peaks from DNA extracted from clots. Molecular genotyping reveals STR loci mismatch in cases 1 through 4 and match in case 5.

Figure 3

XX/XY fluorescence in situ hybridization (FISH) of fetal tissues vs clot areas. FISH analysis of interphase nuclei in formalin-fixed, paraffin-embedded sections using X (green) and Y (red) probes. FISH reveals the predomination of XX cells (arrows). Dotted lines separate fetal tissues from clot.

Figure 4

Histology and immunostains in thrombus (case 4). Thrombus showing lines of Zahn (A, H&E, blue ink marks boundary for short tandem repeat extraction; ×4). Entrapped villi may be a source of contaminating fetal DNA in molecular testing (B, H&E; ×4). The thrombus shows minimal staining for α-fetoprotein (C, ×4; D, ×20), but serum in fetal capillaries is a positive internal control (D, inset). Conversely, immunoglobulin M shows strong staining in the fibrinous portions of the thrombus and negative staining in villi (E, ×4; F, ×20).

Figure 5

Association of clinically detectable fetal maternal hemorrhage (FMH) with intervillous thrombus (IVT). A, FMH volumes are not significantly different between patients with and without IVT. B, Conversely, IVT size is not significantly different between patients with and without FMH. C, Few patients have both IVT size >0 cm and FMH volume >0. D, In those who do, IVT size is nonsignificantly negatively correlated with FMH volume.