Uterine overdistention induces preterm labor mediated by inflammation: observations in pregnant women and nonhuman primates

Abstract

Objective: Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model.

Study design: A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace.

Results: Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an "inflammatory pulse" that correlated with preterm labor (interleukin [IL]-1β, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1β, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth.

Conclusion: Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension.

Keywords: Macaca nemestrina; amniocyte; amnion; chemokine (C-C motif) ligand 2; choriodecidua; cytokines; interleukin-1; interleukin-8; monocyte chemotactic protein 1; myometrium; pregnancy; preterm labor; prostaglandin E2; prostaglandin F2α; tumor necrosis factor; uterine stress; uterine stretch.

FIGURE 1. Temporal relationships among balloon inflation, cytokines and uterine activity

Temporal relationships among intraamniotic balloon inflation, amniotic fluid cytokines, and uterine activity. A, Saline inoculation into amniotic fluid and choriodecidual space days 0 and 4. No balloons were inflated and labor did not occur by day 7. B, Intraamniotic balloon inflation with labor. Two balloons were inflated to maximum balloon inflation of 154 mL over 4 days. “Inflammatory pulse” followed balloon inflation and preceded labor on day 8. C, Intraamniotic balloon inflation without labor. Two balloons were inflated to maximum balloon inflation of 160 mL over 18 days. No labor by day 18. With greater volumes of balloon inflation at end of experiment, there is rise in IL-8. IL, interleukin; PGE₂, prostaglandin E2; PGF_2a, prostaglandin F2_α; TNF, tumor necrosis factor.

FIGURE 2. Messenger RNA (mRNA) levels after amniocyte stretch

Levels of A, IL1, B, TNFα, C, IL6, and D, IL8 mRNA were measured by real-time quantitative reverse transcriptase polymerase chain reaction in amniocytes after 11% stretch (N = 8). Results are corrected for glyceraldehyde 3-phosphate dehydrogenase expression and given as ratio to nonstretched samples (*P < .05). Results are shows as mean with SEM (N = 8). IL, interleukin; TNF, tumor necrosis factor.

FIGURE 3. Messenger RNA (mRNA) levels in amnion from women with polyhydramnios

Levels of A, IL1β, B, TNFα, C, IL6, and D, IL8 mRNA were measured by real-time quantitative reverse transcriptase polymerase chain reaction in amnion cells of women with polyhydramnios (N = 3) vs women delivering preterm not in labor (N = 36). Results are corrected for glyceraldehyde 3-phosphate dehydrogenase expression and are shown as median with range (*P < .05; ***P < .001; P < .0001). Note that there was 1 less amnion sample available from woman with polyhydramnios for this experiment than in Figure 6. IL, interleukin; TNF, tumor necrosis factor.

FIGURE 4. Amnion and myometrium cytokine levels in women with twins

Protein levels of inflammatory cytokines and chemokines in amnion and myometrial tissues of women with twins in early labor vs not in labor (A, C, E, G, and I: amnion; B, D, F, H, and J: myometrium; N = 9, twins in early labor; N = 15, twins not in labor). Results are shown as median with range (*P < .05; **P < .01). CCL, chemokine (C-C motif) ligand; IL, interleukin; TNF, tumor necrosis factor.

FIGURE 5. Quantitative reverse transcriptase PCR validation of differentially expressed myometrial genes

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation of microarray results from nonhuman primate myometrium. X-axis represents individual genes and y-axis, fold-change in expression of difference between balloon inflation cases and saline controls. All genes shown were significant in unadjusted microarray analysis. *Genes significantly up- or down- regulated by qRT-PCR (2-sided t test, P < .05). AGTR1, angiotensin II receptor type 1; CCL2, chemokine (C-C motif) ligand 2; EPHB1, ephrin type-B receptor 1; HBEGF, heparin-binding epidermal growth factor-like growth factor; MME, membrane metalloendopeptidase; TGFB1, transforming growth factor, beta-induced.

FIGURE 6. Quantitative reverse transcriptase PCR on myometrium from women with polyhydramnios and twins

Quantitative reverse transcriptase polymerase chain reaction performed to quantitate messenger RNA levels in myometrium from women with polyhydramnios (N = 4, polyhydramnios; N = 10, singleton gestational age-matched controls not in labor) and twins (N = 9, twins early labor; N = 12, twins not in labor). Levels of AGTR1 (A, polyhydramnios; B, twins), EPHB1 (C, polyhydramnios; D, twins), HBEGF (E, polyhydramnios; F, twins), MME (G, polyhydramnios; H, twins), and TGBI (I, polyhydramnios; J, twins) were measured. Results are shown as median with range (*P < .05; **P < .01). AGTR1, angiotensin II receptor type 1; EPHB1, ephrin type-B receptor 1; HBEGF, heparin-binding EGF-like growth factor; MME, membrane metalloendopeptidase; TGFB1, transforming growth factor, beta-induced.