Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR

Abstract

Human placental villi are surfaced by an outer multinucleated syncytiotrophoblast and underlying mononucleated cytotrophoblasts. Conflicting data have attributed one, or the other, of these villous trophoblast phenotypes to undergo enhanced apoptosis in complicated pregnancies, compared to term, normotensive pregnancies. We use high-resolution confocal microscopy after co-staining for E-cadherin, as a trophoblast plasma membrane marker, and for the cleavage products of cytokeratin 18 and PARP1, as markers for caspase-mediated apoptosis, to distinguish between apoptotic cytotrophoblasts and apoptosis within the syncytiotrophoblast. We test the hypothesis that increased caspase-mediated apoptosis occurs in villi of placentas derived from pregnancies complicated by preeclampsia, intrauterine growth restriction (IUGR), or both. We find significantly elevated apoptosis in villous cytotrophoblasts from women with preeclampsia and/or IUGR, compared to term, normotensive pregnancies. Apoptosis of cytotrophoblasts in villi from complicated pregnancies appears to progress similarly to what we found previously for apoptotic cytotrophoblasts in villi from in term, normotensive pregnancies. Notably, caspase-mediated apoptosis was not detectable in regions with intact syncytiotrophoblast, suggesting strong repression of apoptosis in this trophoblast phenotype in vivo. We suggest that the elevated apoptosis in cytotrophoblasts in preeclampsia contributes to the placental dysfunction characteristic of this disorder. We also propose that repression of apoptosis in the syncytiotrophoblast is important to prevent apoptosis sweeping throughout the syncytium, which would result in widespread death of this essential interface for maternal-fetal exchange.

Fig. 1

Patterns of clCyt18 in apoptotic villous cytotrophoblasts, as detected by immunofluorescence and confocal microscopy. Shown are maximal projections of confocal Z-stacks, with images oriented with the intervillous space to the left. Indistinguishable patterns of clCyt18 were observed in villi from term, normotensive pregnancies, or in pregnancies complicated by preeclampsia, IUGR, or both. clCyt18 was present in cytotrophoblasts as (A) as filamentous structures with stellate processes in cells that contained nuclear DNA, and, (B) as a diffuse staining pattern in cells with intact or fragmented nuclear DNA. These cells were typically round, suggesting detachment from the basement membrane. The most abundant clCyt18 expressing regions in villi from normotensive or complicated pregnancies lacked nuclear DNA but were always outlined by E-cadherin (C–E), suggesting a cytotrophoblast origin. These regions were present as (C) large >5 µm-diameter vesicles or as multiple vesicles of ≤ 5 µm-diameter (D, E; see Supplementary movies 1 and 2). Arrowheads indicate E-cadherin (green) outlining regions expressing clCyt18 (red). Dotted lines indicate apical surface of syncytiotrophoblast, as determined by E-cadherin localization observed in a long exposure.

Fig. 2

E-cadherin and patterns of clCyt18 patterns in placental villi, as detected by immunohistochemistry and light microscopy. Images are oriented with the intervillous space to the left. (A) IgG control (B) E-cadherin staining identifies cytotrophoblasts that are interdigitated into the syncytiotrophoblast (red arrowhead) or underlying the syncytiotrophoblast (white arrowhead). (C, D) Regions of clCyt18 with apparently associated nuclear DNA (blue arrowheads). (E–H) Regions of clCyt18 lacking associated DNA, present as large structures (green arrowheads) or as small structures (black arrowheads).

Fig. 3

clPARP expression and DNA fragmentation in apoptotic cytotrophoblasts. Shown are maximal pixel value projections of confocal Z-stacks, with images oriented with the intervillous space to the left. clPARP was expressed in apoptotic cytotrophoblasts, as identified by co-staining for (A) E-cadherin or (B,C) clCyt18. Insets: (A, B) arrowheads indicate fragmented nuclear DNA. Arrowhead in (C) indicates degraded nuclear DNA in the apoptotic cytotrophoblast. Dotted lines indicate apical surface of syncytiotrophoblast, as determined by E-cadherin localization observed in a long exposure.

Fig. 4

Apoptosis is increased in villous cytotrophoblasts in pregnancies complicated by preeclampsia. Box plot of percent clCyt18-positive cytotrophoblasts in term, normotensive and pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR. *p < 0.05 using the Wilcoxin–Mann–Whitney U test.

Fig. 5

clCyt18 is expressed in regions with fibrin-type fibrinoid. (A–C) Confocal images of tissues stained for E-cadherin, clCyt18 and DNA. Shown are maximal pixel value projections of confocal Z-stacks. (D–F) Light microscopy images of tissues stained by immunohistochemistry for clCyt18 and DNA. Tissues are from the placentas of (A, D) a normotensive patient and patients with (B, E) preeclampsia or (C, F) preeclampsia with IUGR. Dotted lines in (A–C) indicate the surface of the villous, as determined by E-cadherin localization observed in a long exposure. Blue arrowheads in (C) indicated DNA of fragmented nuclei.