Failure of Decidualization and Maternal Immune Tolerance Underlies Uterovascular Resistance in Intra Uterine Growth Restriction

Abstract

Failure of uterine vascular transformation is associated with pregnancy complications including Intra Uterine Growth Restriction (IUGR). The decidua and its immune cell populations play a key role in the earliest stages of this process. Here we investigate the hypothesis that abnormal decidualization and failure of maternal immune tolerance in the second trimester may underlie the uteroplacental pathology of IUGR. Placental bed biopsies were obtained from women undergoing elective caesarian delivery of a healthy term pregnancy, an IUGR pregnancy or a pregnancy complicated by both IUGR and preeclampsia. Decidual tissues were also collected from second trimester terminations from women with either normal or high uterine artery Doppler pulsatile index (PI). Immunohistochemical image analysis and flow cytometry were used to quantify vascular remodeling, decidual leukocytes and decidual status in cases vs. controls. Biopsies from pregnancies complicated by severe IUGR with a high uterine artery pulsatile index (PI) displayed a lack of: myometrial vascular transformation, interstitial, and endovascular extravillous trophoblast (EVT) invasion, and a lower number of maternal leukocytes. Apoptotic mural EVT were observed in association with mature dendritic cells and T cells in the IUGR samples. Second trimester pregnancies with high uterine artery PI displayed a higher incidence of small for gestational age fetuses; a skewed decidual immunology with higher numbers of; CD8 T cells, mature CD83 dendritic cells and lymphatic vessels that were packed with decidual leukocytes. The decidual stromal cells (DSCs) failed to differentiate into the large secretory DSC in these cases, remaining small and cuboidal and expressing lower levels of the nuclear progesterone receptor isoform B, and DSC markers Insulin Growth Factor Binding protein-1 (IGFBP-1) and CD10 as compared to controls. This study shows that defective progesterone mediated decidualization and a hostile maternal immune response against the invading endovascular EVT contribute to the failure of uterovascular remodeling in IUGR pregnancies.

Keywords: EVT; IUGR; T cells; decidua; dendritic; immunology; progesterone; uterovascular transformation.

Figure 1

Failed uterovascular transformation in IUGR is associated with persistance of vascular media and failure of interstitial and endovascular invasion. Immunohistochemical staining was performed to determine the degree of uterine artery transformation in PBBx biopsies using antibodies against smooth muscle actin (SMA, Column 1) cytokeratin 7 (CK7, Column 2) and PECAM (CD31, Column 3). Representative serial sections of control (n = 2/15) and IUGR PBBx (n = 3/8) uterine spiral arteries at the decidual myometrial junction are shown. Asterisks denote the presence of fragmented likely apopototic mural EVT in the IUGR cases. Fibrin deposition in the fully transformed artery is shown in patient 1 Normal marked by the hatched line. Negative control mouse IgG1 is shown in the inset lower right. Scale bars: black = 50 μM, 100x magnification; red = 20 μM, 200x.magnification.

Figure 2

IUGR PBBx display high numbers of preserved uterine spiral arteries. Graphs show quantitation of the degree of remodeling of all myometrial vessels control (circle), IUGR (square) and IUGR with preeclampsia (triangle) groups. Preserved vessels were scored when intact multi layered vascular smooth muscle media was present (A). Disorganized vessels were scored when the vascular media was separated and smooth muscle cells were migrating away from the outer layers of the media (B). Destroyed vessels were scored when all media had been lost and replaced with fibrin (C). *p < 0.05, **p < 0.01.

Figure 3

Failed uterovascular transformation in IUGR is associated with the presence of apoptotic mural EVT. Immunohistochemical staining for cytokeratin 7 (CK7, Column 1) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, Column 2) was perfomed to confirm that the fragmented mural EVT that we observed in the IUGR cases were apoptotic. Representative serial sections of control (n = 2/15) and IUGR PBBx (n = 2/8) uterine spiral arteries at the decidual myometrial junction are shown. Arrows indicate the TUNEL positively nuclei of the mural EVT (mEVT) showing cytokeratin loss in the matching serial sections in the IUGR samples. No TUNEL staining was seen in the interstitial EVT (inEVT) of the IUGR cases or any of the EVT populations of the control term samples. Scale bars: black = 50 μM, 100x magnification; red = 20 μM, 200x.magnification.

Figure 4

Low decidual leukocyte number and increased mature dendritic cells in association with apoptotic EVT in IUGR. Visiopharm Newcast image analysis system was used to define decidual and myometrial regions based on smooth muscle actin staining of the myometrium (A). Representative stained decidual and myometrial sections from normal term PBBx upper panel and IUGR PBBx lower panel (B). Numbers of brown stained CD45+ leukocytes and blue hematoxylin stained decidual stromal cells or myometrial muscle cells were counted in corresponding serial sections across 5% of the respective region using Visiopharm Newcast image analysis system and random meander sampling (C): Normal n = 15, IUGR n = 8, IUGR and PE n = 5, PE n = 8) (^A,Bp < 0.01, ^A−Cp < 0.0001, ^B,Cp < 0.001). Representative serial sections of IUGR PBBx show that the apoptotic mural EVT (CK7, asterisks) that were observed in these biopsies were associated with leukocyte clusters (CD45) containing CD68+ macrophage and elevated number of CD3 T cells and CD83+ mature dendritic cells (D, arrows). Scale bars: black = 500 μM, 20x magnification, blue = 250 μM, 40x magnification, red = 20 μM, 200x.magnification.

Figure 5

Lymphatic vessel density is increased, and leukocytes are abnormally distributed in the high uterine artery PI SGA decidua. Representative serial sections from normal 18 week trimester decidua (top panel), an 18 week high uterine artery PI SGA case (middle panel) and a 18 week high uterine artery case of IUGR (lower panel) stained with antibodies against CK7 (trophoblast) HLAG (EVT), SMA (VSMC), CD31 (endothelial), CD45 (leukocytes) CD56 (uNK) CD68 (macrophage and CD3 (T cells). Lymphatic vessels were identified by staining with D240/podoplanin (lower right 2 images control and IUGR). In the SGA high numbers of small CD31 vessels full of leukocytes were seen (black arrows) In the IUGR decidua this was more pronounced (red arrow), abnormal D240 podoplanin lymphatic endothelial expression was found surrounding the DSC (lower right panel) creating micro-lymphatic vessels that were packed with uNK (CD56) and T cells (CD3). In controls the DSC were negative and only the larger lymphatic vessels stained positive (inset). Scale bars: black = 50 μM, 100x magnification, red= 20μM, 200x magnification.

Figure 6

Elevated cytotoxic CD8⁺ T cells in 2nd trimester high Uterine artery PI and SGA decidua. Multicolored flow cytometry analysis of leukocyte subsets isolated from 2nd trimester normal or high uterine artery PI (>1.6) of decidua. Numbers of CD45⁺ leukocytes, CD56⁺⁺ uNK, and CD68⁺ macrophage were no different between groups (A). CD3+ T cells gated within the CD45⁺ population were doubled in the high uterine artery PI decidua (B). Visiopharm Newcast image analysis system and random meander sampling was used to quantify numbers of CD4⁺ T cells and CD8⁺ T cells in 5% of the total tissue area of each decidual section. Percentages of the total cell number were calculated and are presented as box whisker plots in (C). Representative serial sections from a 15-week normal sample (upper panel) and a 17-week high uterine artery PI SGA) stained with CK7, SMA CD4 and CD8 antibodies are shown in (D) (arrows identify CD8+ T cells). *p < 0.05, **p < 0.01, n = 6 in each group. Scale bars = 20 μM, 200x magnification.

Figure 7

Mature dendritic cells are significantly increased in the 2nd trimester high uterine artery SGA decidua. Representative dot plots of multicolor flow analysis of decidual dendritic cells from an 18-week normal decidua and a decidua from an 18-week high uterine artery PI SGA case. Dendritic differentiation was assessed using three markers CD209/DC-SIGN to mark immature dendritic cells, CD205 to mark intermediate dendritic cells and CD85 to mark mature antigen presenting dendritic cells. Gates were set in the high granular region of the CD45+ gate (A). Comparison across all samples showed that mature CD83+ dendritic cells are increased to 20% of all CD45⁺ leukocytes compared to 4%+- in the controls (B) (p < 0.001, n = 6 per group). Representative immunostained images from one control and 2 high uterine artery PI SGA cases, CD209: immature dendritic cells, CD83: mature antigen presenting dendritic cells (C, arrows indicate positively stained cells). Inset shows rare CD83⁺ dendritic cell in control decidua. Scale bar = 20 μM, 200x magnification.

Figure 8

DSC differentiation fails in the 2nd trimester high uterine artery PI SGA decidua. (A) DSC cell area was measured using the random ellipsoid measurement tool from the Olympus Cellsens software. Immunohistochemical image analysis was performed on serial decidual sections stained with antibodies against the decidual markers PR-B, IGFBP-1, and CD10. Visiopharm Newcast image analysis system and random meander sampling was used to quantify numbers of positively stained DSC in 5% of the total tissue area of each decidual section. Percentages of the total cell number were calculated and are presented as box whisker plots in (A). Representative photographs of serial sections from 2normal 2nd trimester decidua (top panels), and 2 high uterine artery PI SGA cases (lower panels) in each case are shown stained with PR-B (B), IGFBP-1(C) and CD10 (D) antibodies (arrow indicates area of increased lymphatic vessel density). *p < 0.05, **p < 0.01, ***p < 0.0001, n = 6 in each group. Inset shows no staining with the Rabbit IgG negative control. Scale bar = 50 μM, 100x magnification.