Alterations in vaginal microbiome in women with short cervix: longitudinal study of microbial diversity and impact of vaginal progesterone treatment.

OBJECTIVES

A short cervix is a known risk factor for preterm birth, and imbalances in the vaginal microbiome, such as low relative abundance of Lactobacillus, may be associated with an increased risk of preterm birth. The aim of this study was to evaluate differences in the vaginal microbiome between women with a short cervix and those with normal cervical length in the second trimester. Additionally, we aimed to assess longitudinal changes in microbial diversity during pregnancy, as well as the impact of vaginal progesterone treatment on vaginal microenvironment in women with a short cervix.

METHODS

This was a prospective, longitudinal study conducted at Koc University Hospital between January 2020 and May 2023, in women with a singleton pregnancy with a short cervical length (≤ 25 mm) in the second trimester (20 + 0 to 24 + 6 weeks' gestation). After diagnosis of short cervix, administration of 200 mg vaginal progesterone daily was initiated. The control group comprised women with a normal cervical length (> 25 mm) in the second trimester, matched for age and body mass index (BMI). Cervicovaginal swabs were collected from the posterior fornix at three gestational-age ranges: in the first trimester (11 + 0 to 13 + 6 weeks), the second trimester (20 + 0 to 24 + 6 weeks) and the third trimester (28 + 0 to 34 + 6 weeks), and cervical length was measured following sample collection. DNA was extracted and the 16S rRNA bacterial gene was sequenced to analyze and compare the vaginal microbiome between women with a short cervix and controls. We also assessed the microbiome longitudinally in each group, across the first, second and third trimesters. In the short-cervix group, we also compared the microbiome before initiation of progesterone treatment in the second trimester and 4 weeks after its initiation.

RESULTS

Among 490 pregnant women who underwent first-trimester screening during the study period and had vaginal swabs collected, short cervical length was detected in 31 at the second-trimester scan. These women formed the study group. A further 27 women, with a normal cervical length, were matched for BMI and age and assigned to the control group. During the second trimester, women with a short cervix exhibited greater species diversity compared with the control group; this was suggested by the higher Shannon index (0.45 vs 0.33; P = 0.135), which reflects species richness and evenness, and further demonstrated by the higher Chao index (20.2 vs 13.8; P = 0.018), which estimates species richness. In the second trimester, Lactobacillus was less abundant in women with a short cervix than in the control group, although the difference did not reach significance (86.8% vs 95.5%; P = 0.091). At the phylum level, in women with a short cervix compared to those with normal cervical length, the relative abundance of Firmicutes, to which the genus Lactobacillus belongs, was significantly lower (90.7% vs 97.6%; P = 0.041), while the relative abundances of both Bacteroidota (1.73% vs 0.4%; P = 0.004) and Proteobacteria (0.2% vs 0.01%; P = 0.007) were higher. In the second trimester, the relative abundance of Lactobacillus gasseri was significantly lower in women with a short cervix compared to controls (4.7% vs 13.8%; P = 0.023). In the longitudinal analysis of the vaginal microbiome, there were no significant differences among the trimesters in the control group. In contrast, in those with a short cervix, there was a notable decrease in the amount of Lactobacillus crispatus, from 55.0% in the first trimester to 36.1% in the second trimester (P = 0.052). In women with a short cervix, there was no significant difference in bacterial diversity after vs before progesterone treatment (Chao index, 22.6 vs 20.5; P = 0.609).

CONCLUSION

These findings highlight the significant alterations in the vaginal microbiome of pregnant women with a short cervix in comparison to those with normal cervical length, particularly in terms of higher species diversity and distinct community composition. The study also shows that vaginal progesterone treatment in women with a short cervix does not alter the vaginal microbiome, suggesting that it is a safe and effective intervention without disrupting the vaginal microbial balance. Understanding the relationship between cervical length and the vaginal microbiome is essential for developing strategies to reduce the risk of preterm birth in high-risk populations. © 2025 The Author(s). Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.