Embryonic/fetal mortality and intrauterine growth restriction is not exclusive to the CBA/J sub-strain in the CBA × DBA model

Abstract

Inbred strains of mice are powerful models for understanding human pregnancy complications. For example, the exclusive mating of CBA/J females to DBA/2J males increases fetal resorption to 20-35% with an associated decline in placentation and maintenance of maternal Th1 immunity. More recently other complications of pregnancy, IUGR and preeclampsia, have been reported in this model. The aim of this study was to qualify whether the CBA/CaH substrain female can substitute for CBA/J to evoke a phenotype of embryonic/fetal mortality and IUGR. (CBA/CaH × DBA/2J) F1 had significantly higher embryonic/fetal mortality mortality (p = 0.0063), smaller fetuses (p < 0.0001), and greater prevalence of IUGR (<10^th percentile; 47% vs 10%) than (CBA/CaH × Balb/c) F1. Placentae from IUGR fetuses from all mating groups were significantly smaller (p < 0.0001) with evidence of thrombosis and fibrosis when compared to normal-weight fetuses ( > 10^th percentile). In addition, placentae of "normal-weight" (CBA/CaH × DBA/2J) F1 were significantly smaller (p < 0.0006) with a greater proportion of labyrinth (p = 0.0128) and an 11-fold increase in F4/80 + macrophage infiltration (p < 0.0001) when compared to placentae of (CBA/CaH × Balb/c) F1. In conclusion, the embryonic/fetal mortality and IUGR phenotype is not exclusive to CBA/J female mouse, and CBA/CaH females can be substituted to provide a model for the assessment of novel therapeutics.

Figure 1. Macroscopic pregnancy outcomes of murine CBA/CaH × DBA/2J pregnancies.

Representative photographs of gravid uteri harvested from CBA/CaH × Balb/c, CBA/CaH × CBA/CaH and CBA/CaH × DBA/2J pregnancies at (a) 14.5 dpc (N = 20 dams per strain) and (b) 18.5 dpc (N = 30 dams per strain). Fetuses and placentae were excised, measured, weighed and photographed. Weights of fetuses, placentae and resorptions are indicated in grams. Asteri denote resorption sites indicating embryonic/fetal mortality.

Figure 2. Maternal characteristics of the murine CBA/CaH × DBA/2J pregnancies.

(a) Embryonic/fetal mortality (resorption) rate, (b) maternal body weight at mating, (c) uterine wet weight, and (d) number of concepti at 14.5 dpc (N = 20 dams per strain) and 18.5 dpc (N = 30 dams per strain). (e) Representative photograph of asymmetrical diamniotic/dichorionic (placentally fused) twins. All data are represented as mean ± SEM. **p < 0.01 vs value for CBA × Balb/c by One-way ANOVA with Tukey’s multiple comparison test.

Figure 3. Fetal characteristics of the murine CBA/CaH × DBA/2J model.

Fetuses were excised from gravid uteri at 14.5 and 18.5 dpc. (a) Crown-rump length showed a strong positive correlation with fetal wet weight. (b) Distribution of fetal crown-rump length for all strains where a length < 10^th percentile of (CBA × Balb/c) F1 were denoted as intrauterine growth restricted (IUGR; b). ***p = 0.005. ****p < 0.0001 vs (CBA × Balb/c) F1 by One-way ANOVA with Tukey’s multiple comparison test.

Figure 4. Macroscopic and histological characteristics of murine placentae from IUGR and normal-weight fetuses in the CBA/CaH × DBA/2J model.

(a) Representative photograph of IUGR (<10^th %) and normal-weight (>10^th %) littermates at 14.5 dpc (left) and 18.5 dpc (right). (b) Placentae weight for IUGR and normal-weight fetuses represented as mean ± SEM. (c) PAS-stained sections of resorptions and placentae from IUGR fetuses (14.5 dpc) were assessed for differences in placental dimensions and the proportion devoted to the labyrinth, trophospongium and maternal decidua. (d) Comparison of normal labyrinthine tissue and areas of thrombotic (dotted line) and fibrotic tissue (asteri; presence of blue/green collagen) from IUGR associated placentae stained with fast-green Masson’s trichrome. (e) PAS-stained sections of placentae from normal-weight fetuses at 14.5 and 18.5 dpc, and (f) a (CBA/CaH × Balb/c) F1 fetuses at 18.5 dpc placentae that has been colour rendered to show the placental layers quantitated in Table 2. (b) **p < 0.01, ***p < 0.001 vs CBA × CBA by One-way ANOVA with Tukey’s multiple comparison test; ^†p < 0.05 vs value for IUGR by unpaired two-tailed Student T test.

Figure 5. Inflammatory infiltrate of murine placentae from normal-weight fetuses at 14.5 and 18.5 dpc.

(a) Representative micrograph and quantitation of CD3⁺T-lymphocytes in placentae and (b) their distribution among the placental layers. (c) The proportion of CD4⁺ helper T-lymphocytes and CD8⁺ CTLs to the placental T-lymphocyte infiltrate. (d) Representative micrograph and quantitation of F4/80⁺ macrophages and (e) their distribution among the placental layers. In (a–c) micrographs from placentae of (CBA/CaH × Balb/c) F1 fetuses at 18.5 dpc are shown to demonstrate positive staining of the immune cell types. Data are represented as mean ± SEM. N = 34–50 placentae/strain/dpc as outlined in Table 2. **p < 0.01, ****p < 0.0001, as indicated by One-way ANOVA with Tukey’s multiple comparison test.