Trophoblastic oxidative stress in relation to temporal and regional differences in maternal placental blood flow in normal and abnormal early pregnancies

Abstract

Onset of the maternal-placental circulation was studied by Doppler ultrasonography in 65 pairs of age-matched normal and abnormal pregnancies. In normal pregnancies intervillous blood flow increased with gestational age, being detected in 9 of 25 cases at 8 to 9 weeks but in 18 of 20 at 12 to 13 weeks (P = 0.001). By contrast, in abnormal pregnancies flow was detected in nearly all cases (22 of 25) at 8 to 9 weeks (P < 0.001). In addition, regional differences were observed between the groups. Early flow was restricted to the peripheral regions of most normal placentas (P < 0.001), whereas in missed miscarriages it was most common in central regions or throughout the placenta (P < 0.05 and P < 0.001, respectively). Immunoreactivity for heat shock protein 70 and nitrotyrosine residues was greater in samples from peripheral than from central regions of normal placentas (P = 0.028 and P = 0.019, respectively), and from missed miscarriages compared to controls (P = 0.005 and P = 0.001, respectively). Our results indicate that oxidative damage to the trophoblast, induced by premature and widespread onset of the maternal placental circulation secondary to shallow trophoblast invasion, is a key factor in early pregnancy loss. High oxygen concentrations in the periphery of normal early placentas may similarly induce local regression of the villi, leading to formation of the chorion laeve.

Figure 1.

A: Mapping of the placental circulation at 8 weeks of gestation with color Doppler showing the fetal circulation in the umbilical cord (uc) and the maternal circulation within the decidua (d). The dashed lines indicate the boundaries of the regions considered central and peripheral in this example. Note the absence of any signals from within the placenta. cc, chorionic cavity; ac, amniotic cavity. B: Color mapping of the placenta and spectral analysis of the intervillous space at 12 weeks of gestation showing a continuous venous-like flow (arrows) inside the placental tissue.

Figure 2.

A: Light micrograph of a semithin resin section of a placental villus showing marked differences in the staining characteristics with methylene blue of the trophoblast around its circumference. The darkly staining syncytiotrophoblast on the left (single arrow) is considered healthy, whereas the pale staining area on the right (double arrow) is considered stressed. Note that the cytotrophoblast cells under the stressed syncytium are conspicuously elongated, and that they come to the surface at the extreme right-hand margin of the villus (asterisk) where they are forming a new syncytiotrophoblastic layer. B: Higher power photomicrograph of the healthy syncytiotrophoblast at the point marked by a single arrow in A. The apical border of the syncytiotrophoblast (s) carries numerous microvilli, and the syncytioplasm is uniformly dense. The underlying cytotrophoblast cells (arrows) are rounded and evenly spaced along the basement membrane. Both layers of the trophoblast show a similar staining intensity, indicating equivalent protein composition. C: Transmission electron micrograph of an area equivalent to that illustrated in B. Note the apical microvilli, the dense syncytioplasm (s), and the rounded cytotrophoblast cells (c). The syncytial mitochondria (inset) display a regular cristal arrangement. D: Light micrograph of a paraffin section of an 8 week villus demonstrating a healthy syncytiotrophoblast with numerous microvilli and an underlying complete layer of rounded cytotrophoblast cells (arrows). E: Higher power photomicrograph of the stressed area at the point in A marked by the double arrows. Note the absence of microvilli on the apical surface, the vacuolated and leached appearance of the syncytioplasm (s), and the elongated nature of the underlying cytotrophoblast cells (arrows). F: Transmission electron micrograph of an area equivalent to that illustrated in E. There is a complete absence of microvilli, and often the integrity of the apical membrane is lost. The syncytioplasm (s) is heavily vacuolated, and the underlying cytotrophoblast cells (c) are elongated along the basement membrane. The syncytial mitochondria (arrows and inset) display gross dilatation of the intracristal space, similar to that seen in severe oxidative stress in vitro. G: Light micrograph of a paraffin section of an 8 week villus displaying stressed syncytiotrophoblast and flattening of the underlying cytotrophoblast cells (arrows). Scale bars: 500 μm (A); 50 μm (B, E); 5 μm (C, F); 20 μm (D, G).

Figure 3.

Ratio of fluorescent intensity of HSP70 immunoreactivity in the syncytiotrophoblasts from peripheral and central regions of normal placentas at different gestational ages demonstrating that staining was higher in the peripheral regions early in gestation. Although there is considerable variation, reflecting individual differences in timing of the onset of blood flow, there is a general trend for expression to be higher in the peripheral regions of the placenta early in gestation (r = −0.542, P = 0.028). Later in gestation, when blood flow occurs throughout the placenta, the ratio approaches unity.

Figure 4.

Light micrograph of a paraffin section from a 10-week gestational age missed miscarriage in which there was ultrasound evidence of strong intervillous blood flow. The discrepancy between fetal crown rump length and gestational age was nearly 4 weeks, indicating early fetal demise. The syncytiotrophoblast is thinned, with loss of microvilli, and the underlying cytotrophoblast cells are flattened (arrows). The stromal core is avascular, and displays a reduced cellularity. Scale bar, 100 μm.

Figure 5.

Immunohistochemistry for N-Tyr residues in a missed miscarriage at 8 weeks of gestational age (A) and an age-matched normal pregnancy (B), demonstrating the stronger reaction in the former. Staining is particularly intense in the syncytiotrophoblast, but many of the cytotrophoblast cells (arrows) show little immunoreactivity. This pattern is the inverse of that seen for the antioxidant enzymes. Scale bar, 20 μm.

Figure 6.

A: Placenta in situ specimen at 8.5 weeks of gestational age. Villi are present over the entire surface of the chorionic sac, although they are notably shorter over the abembryonic pole in association with the decidua capsularis (asterisk). B: Higher power view of the area indicated by the dashed lines in A illustrating the transition in villous morphology as one moves toward the abembryonic pole. The villi in the lower part, toward the central region of the placenta, are well vascularized, whereas those in the upper part, toward the periphery, contain no blood vessels. Fetal blood vessels can only be traced in the wall of the chorionic sac as far as the arrowhead. C: Higher power view of villi from the lower region of the area illustrated in B showing the presence of fetal vessels in the cellular stromal core. The trophoblast layer is thick, with a well-developed syncytiotrophoblast and a complete layer of underlying cytotrophoblast cells (arrows). There is only a light proteinaceous precipitate in the intervillous space (IVS). D: Higher power view of villi from the upper region of the area illustrated in B confirming the absence of fetal vessels and the reduction in cellularity of the stromal core. The trophoblast covering is thinned, and the syncytiotrophoblast appears to be detaching from the cytotrophoblast cells at certain points (arrows). The cytotrophoblast cells (arrowheads) form an incomplete layer and are flattened. E: Villi from under the decidua capsularis (DC) at the point marked by the asterisk in A. The villi are surrounded by numerous maternal erythrocytes filling the intervillous space (IVS), and display similar trophoblastic changes to those illustrated in D. The villi are equally avascular. F: Resin section of an 11 week villus from the chorion laeve surrounded by maternal erythrocytes. The syncytiotrophoblast (S) is vacuolated, indicating stress, and no cytotrophoblast cells or fetal vessels are present. Scale bars: 1.0 cm (A); 1.0 mm (B); 100 μm (C–E); 20 μm (F).