A recent study published in Scientific Reports describes an impaired progesterone (P4) response in cervical fibroblasts from patients with cervical insufficiency (CI).
Study: Characteristic impairment of progesterone response in cultured cervical fibroblasts obtained from patients with refractory cervical insufficiency. Image Credit: megaflopp / Shutterstock.com
What is cervical insufficiency?
Preterm birth (PTB) impairs perinatal outcomes and is a leading cause of neonatal mortality. CI refers to the inability to sustain pregnancy in the second trimester without labor, clinical contractions, or both.
CI is characterized by premature cervical ripening and membrane prolapse, followed by recurrent pregnancy loss. Although CI most commonly develops after cervical surgery, this condition can also arise in those without surgical history, possibly due to innate abnormalities.
Treatment with P4 receptor (PR) antagonists facilitates cervical ripening in rodents and humans, subsequently resulting in parturition. Previously, researchers have reported the suppression of lipopolysaccharide (LPS)-induced inflammation by P4 in human cervical fibroblasts. Current evidence indicates that P4 helps maintain pregnancy by regulating inflammation, as most P4 receptors are localized in stromal fibroblasts.
About the study
The present study evaluated P4 responsiveness in cervical tissue fibroblasts from patients with refractory CI. Tissue specimens were obtained from pregnant individuals with CI who had a matured cervix, whereas specimens from pregnant individuals without a mature cervix were used as controls. Pregnancies were maintained until 35 gestational weeks with transabdominal cervicoisthmic cerclage (TAC).
Controls underwent elective cesarean sections and had no history of PTB or mid-term miscarriage. Patients with clinical chorioamnionitis, bacterial vaginosis, gestational diabetes, or hypertensive disorder were excluded.
Uterine cervical fibroblasts (UCFs) were precultured with beta-estradiol and incubated with P4 or ethanol. Ribonucleic acid (RNA) was extracted and sequenced for whole-transcriptome analysis.
Study findings
Gene set enrichment analysis of hallmark gene sets from the molecular signatures database revealed no pathophysiological trends between samples from controls and CI subjects in the absence of P4. However, in control UCFs, the pro-inflammatory gene set was suppressed in the presence of P4, whereas the sex hormone response gene set was enhanced.
Comparatively, P4 treatment in CI UCFs enhanced the expression of the pro-inflammatory gene set with no changes in the expression of the sex hormone gene set. Overall, P4 influenced the expression of 387 genes, including 237 downregulated and 150 upregulated genes. A common trend of expression changes in response to P4 was observed in control UCFs but not in CI UCFs.
The expression of genes previously shown to be involved in cervical ripening was not significantly different between CI and control cells, which was confirmed by polymerase chain reaction (PCR) assay. SPARC-like protein 1 (SPARCL1) and FKBP prolyl isomerase 5 (FKBP5) were the representative genes responsive to P4.
Inverted-copy reverse-transcription (RT)-PCR revealed significant upregulation of SPARCL1 and FKBP5 in controls after P4 treatment. By contrast, their expression in CI cells was unaffected.
P4 treatment caused no changes in PR expression in both controls and CI cells. Likewise, cell immunostaining revealed no differences.
A comparative analysis involving real-time PCR, cell immunostaining, and Western blotting of the expression of PR and its two PR-A and PR-B isoforms was performed after P4 treatment. RT-PCR showed no changes in the expression of the PR gene; however, the expression of PR-B was significantly reduced in CI cells.
Western blot analysis did not yield bands corresponding to PR-A or PR-B. Furthermore, PR was stained in controls but not in CI cells.
The inhibitory effects of P4 on LPS-induced inflammation was also explored. To this end, UCFs were stimulated with LPS, followed by P4 treatment, RNA extraction, and RT-PCR.
The inhibitory effect of P4 on LPS induction of prostaglandin-endoperoxide synthase 2 (PTGS2), interleukin-1B (IL-1B), and IL-6 was only evident in controls. Thus, P4-mediated suppression of the LPS-induced inflammatory response was impaired in CI cervical tissues.
Conclusions
A characteristic attenuation of P4-responsiveness was observed in UCFs obtained from pregnant individuals with refractory CI and was accompanied by a marked reduction in PR expression. This may explain the putative mechanisms underlying refractory innate CI.
Although the overall P4 response was significantly diminished in CI cells, residual partial responses were observed. Reports suggest that P4 can also signal through glucocorticoid receptors (GRs).
No significant differences in GR expression were observed between CI cells and controls. Thus, residual partial responses may arise due to crosstalk mediated by GR.
Taken together, the impaired P4 response and reduced PR expression were characteristic of cervical fibroblasts from CI individuals. Future studies on P4-responsive molecules and PR expression regulation are needed to develop clinical strategies for PTB prevention.
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