Anna Paredes Barreda

During gestation, the myometrium remains relaxed but undergoes a phenotypic transformation near term that initiates labor. This coincides with increased expression of contraction-associated proteins (CAPs) and decreased expression of relaxation-associated proteins (RAPs), thereby promoting the development of powerful synchronous contractions that deliver the fetus. We do not yet fully understand the biochemical signaling pathways and physiological changes that occur as human myometrium transitions from a non-laboring to a laboring phenotype. An ex vivo model for the onset of human labor can expand our knowledge. This study aimed to assess whether non-laboring pregnant human myometrium in culture undergoes a phenotypic transformation that is consistent with transitioning toward a labor-like phenotype. Term, non-laboring pregnant human myometrium biopsies were collected. A small portion was immediately snap-frozen (0 h time point), while the remaining tissue was dissected into small pieces, incubated for 48 h in serum-free medium, and then snap-frozen. Changes in the mRNA abundance of key myometrial genes were assessed by qRT-PCR, and protein levels were quantified by Western blotting. Data were analyzed using GraphPad Prism. Myometrial expression of key CAPs, including GJA1 , ESR1 , PTGS2 , NFκB1 , NFκB3 , IL1β , IL6 , and AKR1C1, increased significantly, while expression of the RAPs, PLCL1 , HSPB1 , and HSPB6 , was decreased significantly after 48 h of ex vivo culture. Term, non-laboring pregnant human myometrial explants undergo coordinated molecular remodeling during 48 h of ex vivo culture, with many changes paralleling those reported at term labor in vivo. These findings support prolonged explant culture as a model of the molecular transition toward a labor-like state, while also demonstrating that cultured non-laboring explants may no longer faithfully represent the original non-laboring phenotype.

Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F_2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.

The mechanism by which human labor is initiated in the presence of elevated circulating progesterone levels remains unknown. Recent evidence indicates that the progesterone-metabolizing enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), encoded by the gene AKR1C1, may contribute to functional progesterone withdrawal. We found that AKR1C1 expression significantly increased with labor onset in term myometrium, but not in preterm myometrium. Among preterm laboring deliveries, clinically diagnosed chorioamnionitis was associated with significantly elevated AKR1C1 expression. AKR1C1 expression positively correlated with BMI before labor and negatively correlated with BMI during labor. Analysis by fetal sex showed that AKR1C1 expression was significantly higher in women who delivered male babies compared to women who delivered female babies at term, but not preterm. Further, in pregnancies where the fetus was female, AKR1C1 expression positively correlated with the mother’s age and BMI at the time of delivery. In conclusion, the increase in myometrial AKR1C1 expression with term labor is consistent with 20α-HSD playing a role in local progesterone metabolism to promote birth. Interestingly, this role appears to be specific to term pregnancies where the fetus is male. Upregulated AKR1C1 expression in the myometrium at preterm in-labor with clinical chorioamnionitis suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. The link between AKR1C1 expression and maternal BMI may provide insight into why maternal obesity is often associated with dysfunctional labor. Higher myometrial AKR1C1 expression in male pregnancies may indicate fetal sex-related differences in the mechanisms that precipitate labor onset at term.