Combinatory approaches prevent preterm birth profoundly exacerbated by gene-environment interactions

Abstract

There are currently more than 15 million preterm births each year. We propose that gene-environment interaction is a major contributor to preterm birth. To address this experimentally, we generated a mouse model with uterine deletion of Trp53, which exhibits approximately 50% incidence of spontaneous preterm birth due to premature decidual senescence with increased mTORC1 activity and COX2 signaling. Here we provide evidence that this predisposition provoked preterm birth in 100% of females exposed to a mild inflammatory insult with LPS, revealing the high significance of gene-environment interactions in preterm birth. More intriguingly, preterm birth was rescued in LPS-treated Trp53-deficient mice when they were treated with a combination of rapamycin (mTORC1 inhibitor) and progesterone (P4), without adverse effects on maternal or fetal health. These results provide evidence for the cooperative contributions of two sites of action (decidua and ovary) toward preterm birth. Moreover, a similar signature of decidual senescence with increased mTORC1 and COX2 signaling was observed in women undergoing preterm birth. Collectively, our findings show that superimposition of inflammation on genetic predisposition results in high incidence of preterm birth and suggest that combined treatment with low doses of rapamycin and P4 may help reduce the incidence of preterm birth in high-risk women.

Figure 1. Mild inflammatory insult in p53^d/d female mice upregulates decidual COX2 signaling and renders ovaries more sensitive to luteolysis.

(A) Immunohistochemistry showed upregulated COX2 expression in the decidua of Trp53^loxP/loxPPgr^Cre/+ (p53^d/d) mice 12 hours after an injection of 10 μg LPS at 1900h on day 16 of pregnancy. Dec, decidua; Sp, spongiotrophoblast; Lb, labyrinth. Scale bar: 250 μm. (B) Mass spectrometric analysis showed that uterine levels of PGF2α, but not PGE2, are significantly upregulated in LPS-treated p53^d/d females as compared with Trp53^loxP/loxPPgr^+/+ (p53^fl/fl) littermates. This upregulation was suppressed by celecoxib treatment. Three to 6 independent samples isolated from each mouse were analyzed (n = 3–5 mice/treatment group; mean ± SEM; *P < 0.05). (C) Serum P₄ levels were measured 12 hours after LPS or vehicle injection. p53^d/d females showed significant decreases in serum P₄ levels as compared with p53^fl/fl littermates, which did not show any significant differences (mean ± SEM; *P < 0.05). (D) qPCR results showed significant upregulation of Akr1c18 in ovaries of p53^d/d females after LPS injection compared with those in p53^fl/fl littermates. This upregulation was attenuated by rapamycin (Rapa) and P₄ treatment (mean ± SEM). (E) Immunohistochemistry for 20αHSD in CL of vehicle-treated p53^fl/fl and p53^d/d females showed similar signal levels, as compared with increased signal levels in CL of p53^d/d females 12 hours after LPS injection, albeit with some increases in p53^fl/fl CL. As expected, sections of ovaries from p53^fl/fl females on day 20 of pregnancy prior to parturition showed higher expression of 20αHSD (positive control). Scale bars: 100 μm.

Figure 2. Expression of Prl3c1 and Prlr is downregulated in decidua and at the decidual-placental interface in p53^d/d females.

(A) In situ hybridization of Prl3c1 in p53^fl/fl and p53^d/d deciduae on day 8 of pregnancy showing significant downregulation in p53^d/d decidua. Scale bar: 1 mm. (B) In situ hybridization of Prl3c1 and Prlr in p53^d/d and p53^fl/fl deciduae on day 16 of pregnancy, again showing downregulation in p53^d/d females. Dotted lines demarcate the location of trophoblast giant cells (TGC) at the decidual-placental interface. Scale bar for Prl3c1: 1 mm; for Prlr: 500 μm. Emb, embryo; AM, antimesometrial pole; M, mesometrial pole; Lb, labyrinth.

Figure 3. Preterm birth in p53^d/d females was effectively rescued with combined treatment of rapamycin and P₄, without adverse effects on pregnancy outcome.

(A) All p53^d/d females examined under mild inflammation (10 μg LPS) showed preterm birth, which was rescued by a combination of rapamycin (Rapa) and P₄ treatment with or without celecoxib (mean ± SEM; *P < 0.05 compared with vehicle-treated control females; **P < 0.05 compared with LPS-treated females). (B) p53^d/d females with these treatment schedules showed rescue of preterm birth as assessed by the day of delivery (mean ± SEM; *P < 0.05). (C) Combined treatment with rapamycin, P₄, and celecoxib adversely affects fetal health in p53^fl/fl females, but treatment with P₄ and rapamycin does not. a, b, and c denote dead pups/resorption sites in one dam in each group (see Supplemental Table 2). (D) Immunohistochemistry for COX2 in deciduae of LPS-treated p53^d/d females showed decreased signals after treatment with rapamycin and P₄. Scale bar: 200 μm. (E) Mass spectrometric analysis of PGs shows that treatment with rapamycin and P₄ significantly lowered PGF_2α levels in p53^d/d uteri challenged with LPS; uterine PGE₂ levels were not significantly different in similarly treated p53^fl/fl and p53^d/d females. Three to 6 independent samples isolated per animal were analyzed (n = 3–5 mice/treatment group; mean ± SEM; *P < 0.05). Veh, vehicle.

Figure 4. Heightened decidual senescence, mTORC1 signaling, and COX2 levels are evident in human preterm birth.

(A) Three representative images of increased SA-β-gal staining (blue) in sections of preterm decidual-placental samples compared with those from term delivery. (B) Two representative images showing increased signals for γH2AX, a senescence and DNA damage response marker, in preterm decidual-placental sections compared with those from term delivery. Immunostaining for pS6 (C) and COX2 (D) in sections of preterm decidual-placental samples compared with those from term delivery. The same 9 samples were processed as paraffin sections for COX2, pS6, and γH2AX immunostaining, while 9 additional samples were processed as frozen sections for SA-β-gal staining. Dec, decidua; Vil, villous trophoblast. Scale bars: 200 μm.

Figure 5. Levels of IL-6 and IL-8 and expression of PTGS2 and AKR1C1 in cultured human term decidual cells following exposure to LPS.

(A and B) Decidual cells isolated from human term placentae showed increased secretion of IL-6 and IL-8 into culture media when exposed to LPS for 24 hours in culture; the levels were attenuated by treatment with rapamycin and/or P₄ (mean ± SEM; *P < 0.05 compared with vehicle-treated control; **P < 0.05 compared with LPS-treated cells). ELISA for IL-6 (n = 8) and IL-8 (n = 7) were repeated using independent samples. (C) qPCR results showed dose-dependent increases in decidual PTGS2 expression levels 6 hours after LPS exposure. Experiments were repeated in 3 independent samples (mean ± SEM; *P < 0.05 compared with control). (D) Western blotting showed increases in decidual COX2 levels. One representative blot from 3 independent experiments is shown. (E) qPCR showed dose-dependent increases in decidual AKR1C1 expression levels 6 hours after LPS exposure. Experiments were repeated in 3 independent samples (mean ± SEM; *P < 0.05 compared with control).

Figure 6. Proposed scheme of gene-environment interactions in preterm birth.

In mice with uterine deletion of Trp53, premature decidual senescence arising from heightened decidual mTORC1 and COX2 signaling confers genetic predisposition to preterm birth. This genetic predisposition is remarkably aggravated by a mild inflammatory insult through a decrease in ovarian P₄ levels due to increased expression of 20αHSD, a P₄ metabolizing enzyme. Decidua-derived factors normally serve as luteotrophins to extend the CL lifespan; decidual health is presumably compromised in Trp53^loxP/loxPPgr^Cre/+ females due to premature senescence and reduced levels of decidual factors, conferring ovarian insufficiency and increased susceptibility to inflammation-mediated preterm birth.