Early first trimester uteroplacental flow and the progressive disintegration of spiral artery plugs: new insights from contrast-enhanced ultrasound and tissue histopathology

Abstract

Study question: Does the use of a vascular contrast agent facilitate earlier detection of maternal flow to the placental intervillous space (IVS) in the first trimester of pregnancy?

Summary answer: Microvascular filling of the IVS was demonstrated by contrast-enhanced ultrasound from 6 weeks of gestation onwards, earlier than previously believed.

What is known already: During placental establishment and remodeling of maternal spiral arteries, endovascular trophoblast cells invade and accumulate in the lumen of these vessels to form 'trophoblast plugs'. Prior evidence from morphological and Doppler ultrasound studies has been conflicting as to whether the spiral arteries are completely plugged, preventing maternal blood flow to the IVS until late in the first trimester.

Study design, size, duration: Uteroplacental flow was examined across the first trimester in human subjects given an intravenous infusion of lipid-shelled octofluoropropane microbubbles with ultrasound measurement of destruction and replenishment kinetics. We also performed a comprehensive histopathological correlation using two separately archived uteroplacental tissue collections to evaluate the degree of spiral artery plugging and evaluate remodeling of the upstream myometrial radial and arcurate arteries.

Participants/materials, setting, methods: Pregnant women (n = 34) were recruited in the first trimester (range: 6+3 to 13+6 weeks gestation) for contrast-enhanced ultrasound studies with destruction-replenishment analysis of signal intensity for assessment of microvascular flux rate. Histological samples from archived in situ (Boyd Collection, n = 11) and fresh first, second, and third trimester decidual and post-hysterectomy uterine specimens (n = 16) were evaluated by immunohistochemistry (using markers of epithelial, endothelial and T-cells, as well as cell adhesion and proliferation) and ultrastructural analysis.

Main results and the role of chance: Contrast agent entry into the IVS was visualized as early as 6+3 weeks of gestation with some variability in microvascular flux rate noted in the 6-7+6 week samples. Spiral artery plug canalization was observed from 7 weeks with progressive disintegration thereafter. Of note, microvascular flux rate did not progressively increase until 13 weeks, which suggests that resistance to maternal flow in the early placenta may be mediated more proximally by myometrial radial arteries that begin remodeling at the end of the first trimester.

Limitations reasons for caution: Gestational age was determined by crown-rump length measurements obtained by transvaginal ultrasound on the day of contrast-enhanced imaging studies, which may explain the variability in the earliest gestational age samples due to the margin of error in this type of measurement.

Wider implications of the findings: Our comprehensive in situ histological analysis, in combination with the use of an in vivo imaging modality that has the sensitivity to permit visualization of microvascular filling, has allowed us to reveal new evidence in support of increasing blood flow to the IVS from 6 weeks of gestation. Histologic review suggested the mechanism may be blood flow through capillary-sized channels that form through the loosely cohesive 'plugs' by 7 weeks gestation. However, spiral artery remodeling on its own did not appear to explain why there is significantly more blood flow at 13 weeks gestation. Histologic studies suggest it may be related to radial artery remodeling, which begins at the end of the first trimester.

Study funding/competing interest(s): This project was supported by the Oregon Health and Science University Knight Cardiovascular Institute, Center for Developmental Health and the Struble Foundation. There are no competing interests.

Keywords: Spiral artery; contrast-enhanced ultrasound; in vivo imaging; intervillous blood flow; placental perfusion; trophoblast plugs.

Figure 1

Contrast-enhanced ultrasound flux rate and variance. (A) Example replenishment kinetic curve demonstrating calculation of flux rate (β) from the slope of the curve (y). (B) Flux rate (β value in sec^-1) plotted over 6–13 weeks of human gestation (mean + SEM). The number of participants is represented by the number overlay in each bar. The variance between measures of flux rate within (C) individual data acquisition replicates and (D) in two different anatomical orientations.

Figure 2

Characterization of channels within the spiral artery plugs. (A) En bloc sections from the Boyd Collection (gross example is from 10 weeks’ gestation) were available for serial section analysis revealing well-formed channels (*) through loosely cohesive endovascular trophoblast cells (B), which trichrome staining suggested may be related to cell death at the maternal arterial blood interface (arrow). (C) Elective termination specimens were employed for immunophenotyping (H&E: hematoxylin and eosin). (D) EVT cells expressed less E-cadherin (cell adhesion marker) than the strong staining seen in villous cytotrophoblast cells (top left). (E) The endovascular plugs had their own specific CD56 (cell adhesion marker: also stains uterine natural killer cells) staining pattern, which was absent in all other types of trophoblast. (F) Although the overlying anchoring villi were proliferating, the cells making up the spiral artery plugs were Ki-67 (proliferation marker) negative. Figure 2B was photographed using a 20× objective (~200×); C–F were photographed using a 10× objective (~100× magnification).

Figure 3

Analysis of vascular channels through spiral artery plugs. (A) By 8 weeks gestation the channels were well-formed and clearly communicated with the intervillous space. (B) They were lined by trophoblast cells negative for CD31 (endothelial marker, *). (C–F) Electron microscopy showed loosely cohesive cells connected by desmosomes (red arrow) that were undergoing necrotic degeneration forming fibrin-lined channels. A and B were photographed using a 20× objective (~200× magnification). C and D 3000× (note RBCs); E 6000× (note RBCs); F 60 000×.

Figure 4

Pregnancy-induced remodeling of myometrial radial and arcuate arteries. (A) The radial arteries appeared to begin remodeling at the end of the first trimester associated with perivascular lymphocytes and few EVT cells that track along the adventitia, but they did not invade the media. (B) By 15 weeks the upstream proximal radial arteries showed signs of remodeling that was clearly evident in the 33-week hysterectomy specimen (C) and left permanent fibrin changes seen in multigravida postmenopausal specimens (D). Compared with nulligravida uteri (E), the arcuate arteries also dilated and attenuated (F); this occurred in the absence of trophoblast. All sections stained with H&E and photographs taken using a 5× objective (~50× magnification).

Figure 5

Radial artery remodeling involves perivascular trophoblasts and accumulation of medial CD3 positive and CD3 negative lymphocytes. (A) Remodeling radial artery at 15 weeks gestation by H&E stain showed medial muscular and extracellular matrix changes appreciated best in complete cross-sections (dashed circle). (B) Periodic acid–Schiff stain highlighted pervivascular adentitia that was invaded by cytokeratin positive EVT cells (C) that do not appear to invade the muscular media. (D) Instead, there are numerous lymphocytes, including CD3-positive T-cells in the walls of these remodeling arteries. Photographs taken using a 5× objective (~50× magnification).