Prostaglandins are essential for cervical ripening in LPS-mediated preterm birth but not term or antiprogestin-driven preterm ripening

Abstract

Globally, an estimated 13 million preterm babies are born each year. These babies are at increased risk of infant mortality and life-long health complications. Interventions to prevent preterm birth (PTB) require an understanding of processes driving parturition. Prostaglandins (PGs) have diverse functions in parturition, including regulation of uterine contractility and tissue remodeling. Our studies on cervical remodeling in mice suggest that although local synthesis of PGs are not increased in term ripening, transcripts encoding PG-endoperoxide synthase 2 (Ptgs2) are induced in lipopolysaccharide (LPS)-mediated premature ripening. This study provides evidence for two distinct pathways of cervical ripening: one dependent on PGs derived from paracrine or endocrine sources and the other independent of PG actions. Cervical PG levels are increased in LPS-treated mice, a model of infection-mediated PTB, consistent with increases in PG synthesizing enzymes and reduction in PG-metabolizing enzymes. Administration of SC-236, a PTGS2 inhibitor, along with LPS attenuated cervical softening, consistent with the essential role of PGs in LPS-induced ripening. In contrast, during term and preterm ripening mediated by the antiprogestin, mifepristone, cervical PG levels, and expression of PG synthetic and catabolic enzymes did not change in a manner that supports a role for PGs. These findings in mice, supported by correlative studies in women, suggest PGs do not regulate all aspects of the parturition process. Additionally, it suggests a need to refocus current strategies toward developing therapies for the prevention of PTB that target early, pathway-specific processes rather than focusing on common late end point mediators of PTB.

Figure 1.

Cervical expression for PTGS1 and PTGS2 mRNA and protein remains low to undetectable throughout pregnancy. The mRNA expression of Ptgs1 (A) and Ptgs2 (B) in the nonpregnant (NP), gestational days 8–18. and postpartum (PP) cervix. Data indicate average ± SEM (n = 4–6). *, Significance (P ≤ .05) compared with gestational day 18. C, Protein expression of PTGS1 at time points between gestational days 6–18 and 1 day postpartum. Gestational day 18 uterus from Ptgs1 knockout (KO) mice is a negative control, whereas the wild-type (WT) day 18 uterus serves as a positive control. D, Protein expression of PTGS2 at time points between gestational days 6–18 and 1 day postpartum. As a positive control, LPS-treated uterine tissue from a NP WT mouse was evaluated. Anti-PTGS2 recognizes two closely migrating bands. The larger-size band at 75 kDa is the specific band because it is not present in the Ptgs2 KO cervix (see Figure 2A). Anti-α-tubulin is the loading control. These Western blots are a representative image from one of three experiments. Densitometric quantitation of three protein blots is indicated in panel C and panel D. The positive control was not included in the statistical analysis. *, Significance (P ≤ .05) compared with gestational day 18.

Figure 2.

Cervical PTGS2 but not PTGS1 protein expression is induced in LPS-mediated cervical ripening. A, From left to right, cervical protein from gestational day 15 (NT), gestational day 15 ± mifepristone (MFP), day 15 ± IU-LPS, gestational day 18, and 2 hours PP. As a negative and positive control, respectively, in the upper panel, uterine protein (Ut) from nonpregnant (NP) Ptgs1 null (Ptgs1 KO Ut) and gestational day 18 wild-type (d18 Ut), and lower panel, uterine protein from Ptgs2 null mice treated with LPS (Ptgs2 KO NP LPS Ut) and LPS-treated wild-type mice (NP LPS Ut). α-Tubulin is the loading control. Blots are representative of three experiments each. Densitometric quantitation of three protein blots is indicated below each blot. *, Significance (P ≤ .05) compared with NT. The ± controls were not included in the statistical analysis. B, Immunohistochemical detection of PTGS2-expressing cells in cervical sections collected at gestational day 15 (untreated), day 15 mifepristone, day 15 IU-LPS at a ×100 magnification and day 15 IU-LPS at a ×200 magnification. Positive staining is indicated by a brownish red stain. Stroma (S), basal epithelia (BE), and endothelial cells surrounding the blood vessels (EN) are shown. Each image is representative of three animals for each treatment.

Figure 3.

LPS treatment promotes prostaglandin synthesis. A, Cervical mRNA expression of prostaglandin E synthase (Ptges) is increased and 15-hydroxyprostaglandin dehydrogenase (Pgdh) is decreased in LPS-mediated ripening but not term or mifepristone-mediated ripening. Gestational day 15, no treatment (NT), day 15 mifepristone (MFP), day 15 IU-LPS, gestational day 18 (d18), 2 and 24 hours postpartum (PP) are shown. Data represent the average of four to six cervices for each time point or treatment ± SEM. *, Significance (P ≤ .05) compared with untreated day 15 cervices. B, Cervical tissue prostaglandin levels are elevated in LPS-mediated but not mifepristone-mediated preterm ripening or term ripening. Prostaglandin levels measured at gestational day 15 with no treatment (NT), day 15 mifepristone (MFP), day 15 IU-LPS, gestational day 18 (d18), and 24 hours PP are shown. Data represent an average of three pools of cervical tissue for each time point or treatment group ± SEM. Each pool consisted of 4–10 cervices. *, Significance (P ≤ .05) compared with untreated day 15 cervices. 6-KGF_1a, 6-ketoprostaglandin F_1a.

Figure 4.

The level of cervical transcripts encoding PGE₂ receptors (Ptger1–4) is not influenced by LPS treatment. Gestational day 15 (NT), day 15 mifepristone (MFP), day 15 IU-LPS, gestational day 18 (d18), 2 and 24 hours PP are shown. Data represent the average of four to six cervices for each time point or treatment ± SEM. *, Significance (P ≤ .05) when compared with untreated day 15 cervices.

Figure 5.

Plasma progesterone levels decline yet estrogen levels do not increase in mice treated with LPS ± prostaglandin synthesis inhibitor to the same degree as observed in physiological cervical ripening at term. A, Plasma concentrations of progesterone at gestational day 15 (NT), day 15 mifepristone (MFP), day 15 12-hour IP-LPS, day 15 12-hour IP-LPS + SC-236, day 15 6-hour IU-LPS, and gestational day 18 (d18). B, Plasma estradiol levels as described in panel A. Steroid data represent the average of 6–10 animals ± SEM. *, Significance (P ≤ .05) compared with day 15 untreated; +, significance (P ≤ .05) compared with day 18. C, Progesterone to estrogen (P:E) ratios in preterm models of infection (LPS) and hormone withdrawal (mifepristone).

Figure 6.

Blocking PTGS2 results in a decreased expression of proinflammatory molecules and a decrease in neutrophil recruitment. A, The mRNA expression of Il6, Tnf, Cxcl2, and Mmp8 in cervices of mice 6 and/or 12 hours after ip LPS injection. Data represent the average of six to seven cervices ± SEM. *, Significance (P ≤ .05) compared with untreated day 15 cervices. B, Flow cytometry was used to determine how SC-236 affects the recruitment of neutrophils to the cervix. The pan leukocyte marker CD45 was used to sort cells, and the neutrophil population was defined as GR1 positive and Neu 7/4 high expressing cells. C, SC-236 treatment results in a significant decline in the recruitment of neutrophils, although not to the level observed in the untreated day 15 cervices. The data represent four to eight animals ± SEM. Neu, neutrophils. *, Significance when compared with untreated day 15 cervices; †, significance compared with IP-LPS-treated cervices (P ≤ .05).

Figure 7.

Prostaglandins are sufficient to promote cervical ripening and are necessary for LPS-induced premature cervical ripening. A, Biomechanical testing of cervices obtained at gestational day 15 (d15), day 18 (d18), day 15 mifepristone (MFP), day 15 6-hour IU-LPS (IU LPS), day 15 12-hour IP-LPS (IP LPS), day 15 12-hour IP-LPS + SC-236, and day 15 6-hour misoprostol (Miso) [n = 17 (d15); n = 6 (d18); n = 5 (MFP); n = 9 (IU LPS); n = 3 (IP LPS); n = 5 (IP LPS+SC-236); and n = 5 (d15 misoprostol)]. Groups within a single bracket are not significantly different from each other, whereas each bracketed group is significantly different (P ≤ .05) from every other bracketed group. B, Table showing coefficient estimates (mean ± SEM) for all groups in panel A. *, Rows with the same numeric indicator are not judged statistically different.