OBJECTIVE: To determine the association between microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI) and the cervical prevalence of Gardnerella vaginalis DNA in pregnancies with preterm prelabor rupture of membrane (PPROM). METHOD: In total, 405 women with singleton pregnancies complicated with PPROM were included. Cervical fluid and amniotic fluid samples were collected at the time of admission. Bacterial and G. vaginalis DNA were assessed in the cervical fluid samples using quantitative PCR technique. Concentrations of interleukin-6 and MIAC were evaluated in the amniotic fluid samples. Loads of G. vaginalis DNA ≥ 1% of the total cervical bacterial DNA were used to define the cervical prevalence of G. vaginalis as abundant. Based on the MIAC and IAI, women were categorized into four groups: with intra-amniotic infection (both MIAC and IAI), with sterile IAI (IAI without MIAC), with MIAC without IAI, and without either MIAC or IAI. RESULTS: The presence of the abundant cervical G. vaginalis was related to MIAC (with: 65% vs. without: 44%; p = 0.0004) but not IAI (with: 52% vs. without: 48%; p = 0.70). Women with MIAC without IAI had the highest load of the cervical G. vaginalis DNA (median 2.0 × 104 copies DNA/mL) and the highest presence of abundant cervical G. vaginalis (73%). CONCLUSIONS: In women with PPROM, the presence of cervical G. vaginalis was associated with MIAC, mainly without the concurrent presence of IAI.

Biomedical Research Center University Hospital Hradec Kralove Hradec Kralove Czech Republic

Department of Genetics and Bioinformatics Domain of Health Data and Digitalisation Institute of Public Health Oslo Norway

Department of Obstetrics and Gynecology Institute of Clinical Science Sahlgrenska Academy Gothenburg University Gothenburg Sweden

Department of Obstetrics and Gynecology Region Västra Götaland Sahlgrenska University Hospital Gothenburg Sweden

Department of Obstetrics and Gynecology University Hospital Brno Faculty of Medicine Masaryk University Brno Czech Republic

Department of Obstetrics and Gynecology University Hospital Hradec Kralove Charles University Faculty of Medicine in Hradec Kralove Hradec Kralove Czech Republic

Department of Obstetrics and Gynecology University Hospital Ostrava Ostrava Czech Republic

Institute of Clinical Biochemistry and Diagnostics University Hospital Hradec Kralove Hradec Kralove Czech Republic

See more in PubMed

Mercer BM. Preterm premature rupture of the membranes. Obstet Gynecol. 2003;101(1):178–93. 10.1016/s0029-7844(02)02366-9 . PubMed DOI

Mercer BM. Preterm premature rupture of the membranes: current approaches to evaluation and management. Obstet Gynecol Clin North Am. 2005;32(3):411–28. 10.1016/j.ogc.2005.03.003 . PubMed DOI

Shubert PJ, Diss E, Iams JD. Etiology of preterm premature rupture of membranes. Obstet Gynecol Clin North Am. 1992;19(2):251–63. . PubMed

Lee T, Silver H. Etiology and epidemiology of preterm premature rupture of the membranes. Clin Perinatol. 2001;28(4):721–34. 10.1016/s0095-5108(03)00073-3 . PubMed DOI

Menon R, Richardson LS. Preterm prelabor rupture of the membranes: A disease of the fetal membranes. Semin Perinatol. 2017;41(7):409–19. 10.1053/j.semperi.2017.07.012 PubMed DOI PMC

Kumar D, Moore RM, Mercer BM, Mansour JM, Redline RW, Moore JJ. The physiology of fetal membrane weakening and rupture: Insights gained from the determination of physical properties revisited. Placenta. 2016;42:59–73. 10.1016/j.placenta.2016.03.015 . PubMed DOI

Musilova I, Kutova R, Pliskova L, Stepan M, Menon R, Jacobsson B, et al. Intraamniotic Inflammation in Women with Preterm Prelabor Rupture of Membranes. PLoS One. 2015;10(7):e0133929 10.1371/journal.pone.0133929 PubMed DOI PMC

Musilova I, Kacerovsky M, Stepan M, Bestvina T, Pliskova L, Zednikova B, et al. Maternal serum C-reactive protein concentration and intra-amniotic inflammation in women with preterm prelabor rupture of membranes. PLoS One. 2017;12(8):e0182731 10.1371/journal.pone.0182731 PubMed DOI PMC

Musilova I, Pliskova L, Gerychova R, Janku P, Simetka O, Matlak P, et al. Maternal white blood cell count cannot identify the presence of microbial invasion of the amniotic cavity or intra-amniotic inflammation in women with preterm prelabor rupture of membranes. PLoS One. 2017;12(12):e0189394 10.1371/journal.pone.0189394 PubMed DOI PMC

Kroon SJ, Ravel J, Huston WM. Cervicovaginal microbiota, women's health, and reproductive outcomes. Fertil Steril. 2018;110(3):327–36. 10.1016/j.fertnstert.2018.06.036 . PubMed DOI

Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A. 2011;108 Suppl 1:4680–7. 10.1073/pnas.1002611107 PubMed DOI PMC

Anton L, Sierra LJ, DeVine A, Barila G, Heiser L, Brown AG, et al. Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health. Front Microbiol. 2018;9:2181 10.3389/fmicb.2018.02181 PubMed DOI PMC

Rampersaud R, Planet PJ, Randis TM, Kulkarni R, Aguilar JL, Lehrer RI, et al. Inerolysin, a cholesterol-dependent cytolysin produced by Lactobacillus iners. J Bacteriol. 2011;193(5):1034–41. 10.1128/JB.00694-10 PubMed DOI PMC

Kacerovsky M, Pliskova L, Bolehovska R, Gerychova R, Janku P, Matlak P, et al. Lactobacilli-dominated cervical microbiota in women with preterm prelabor rupture of membranes. Pediatr Res. 2019. 10.1038/s41390-019-0692-1 . PubMed DOI

Kacerovsky M, Vrbacky F, Kutova R, Pliskova L, Andrys C, Musilova I, et al. Cervical microbiota in women with preterm prelabor rupture of membranes. PLoS One. 2015;10(5):e0126884 10.1371/journal.pone.0126884 PubMed DOI PMC

Spiegel CA, Davick P, Totten PA, Chen KC, Eschenbach DA, Amsel R, et al. Gardnerella vaginalis and anaerobic bacteria in the etiology of bacterial (nonspecific) vaginosis. Scand J Infect Dis Suppl. 1983;40:41–6. . PubMed

Machado A, Cerca N. Influence of Biofilm Formation by Gardnerella vaginalis and Other Anaerobes on Bacterial Vaginosis. J Infect Dis. 2015;212(12):1856–61. 10.1093/infdis/jiv338 . PubMed DOI

Hyman RW, Fukushima M, Diamond L, Kumm J, Giudice LC, Davis RW. Microbes on the human vaginal epithelium. Proc Natl Acad Sci U S A. 2005;102(22):7952–7. 10.1073/pnas.0503236102 PubMed DOI PMC

Mikamo H, Sato Y, Hayasaki Y, Kawazoe K, Hua YX, Tamaya T. Bacterial isolates from patients with preterm labor with and without preterm rupture of the fetal membranes. Infect Dis Obstet Gynecol. 1999;7(4):190–4. 10.1155/S1064744999000320 PubMed DOI PMC

Shimaoka M, Yo Y, Doh K, Kotani Y, Suzuki A, Tsuji I, et al. Association between preterm delivery and bacterial vaginosis with or without treatment. Sci Rep. 2019;9(1):509 10.1038/s41598-018-36964-2 PubMed DOI PMC

Romero R, Mazor M, Wu YK, Sirtori M, Oyarzun E, Mitchell MD, et al. Infection in the pathogenesis of preterm labor. Semin Perinatol. 1988;12(4):262–79. . PubMed

Musilova I, Andrys C, Holeckova M, Kolarova V, Pliskova L, Drahosova M, et al. Interleukin-6 measured using the automated electrochemiluminescence immunoassay method for the identification of intra-amniotic inflammation in preterm prelabor rupture of membranes. J Matern Fetal Neonatal Med. 2018:1–131. 10.1080/14767058.2018.1533947 . PubMed DOI

Liu CM, Aziz M, Kachur S, Hsueh PR, Huang YT, Keim P, et al. BactQuant: an enhanced broad-coverage bacterial quantitative real-time PCR assay. BMC Microbiol. 2012;12:56 10.1186/1471-2180-12-56 PubMed DOI PMC

Fouhy F, Deane J, Rea MC, O'Sullivan O, Ross RP, O'Callaghan G, et al. The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. PloS one. 2015;10(3):e0119355 10.1371/journal.pone.0119355 PubMed DOI PMC

Kacerovsky M, Musilova I, Hornychova H, Kutova R, Pliskova L, Kostal M, et al. Bedside assessment of amniotic fluid interleukin-6 in preterm prelabor rupture of membranes. Am J Obstet Gynecol. 2014;211(4):385 e1–9. 10.1016/j.ajog.2014.03.069 . PubMed DOI

Benjamino J, Lincoln S, Srivastava R, Graf J. Low-abundant bacteria drive compositional changes in the gut microbiota after dietary alteration. Microbiome. 2018;6(1):86 10.1186/s40168-018-0469-5 PubMed DOI PMC

Stewart CJ, Embleton ND, Marrs ECL, Smith DP, Fofanova T, Nelson A, et al. Longitudinal development of the gut microbiome and metabolome in preterm neonates with late onset sepsis and healthy controls. Microbiome. 2017;5(1):75 Epub 2017/07/14. 10.1186/s40168-017-0295-1 PubMed DOI PMC

Shang Y, Khafipour E, Derakhshani H, Sarna LK, Woo CW, Siow YL, et al. Short Term High Fat Diet Induces Obesity-Enhancing Changes in Mouse Gut Microbiota That are Partially Reversed by Cessation of the High Fat Diet. Lipids. 2017;52(6):499–511. Epub 2017/04/22. 10.1007/s11745-017-4253-2 . PubMed DOI

Depner M, Ege MJ, Cox MJ, Dwyer S, Walker AW, Birzele LT, et al. Bacterial microbiota of the upper respiratory tract and childhood asthma. J Allergy Clin Immunol. 2017;139(3):826–34 e13. Epub 2016/09/01. 10.1016/j.jaci.2016.05.050 . PubMed DOI

DiGiulio DB, Romero R, Kusanovic JP, Gomez R, Kim CJ, Seok KS, et al. Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes. Am J Reprod Immunol. 2010;64(1):38–57. 10.1111/j.1600-0897.2010.00830.x PubMed DOI PMC

Kacerovsky M, Celec P, Vlkova B, Skogstrand K, Hougaard DM, Cobo T, et al. Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria. PLoS One. 2013;8(3):e60399 10.1371/journal.pone.0060399 PubMed DOI PMC

Chaemsaithong P, Romero R, Korzeniewski SJ, Martinez-Varea A, Dong Z, Yoon BH, et al. A rapid interleukin-6 bedside test for the identification of intra-amniotic inflammation in preterm labor with intact membranes. J Matern Fetal Neonatal Med. 2016;29(3):349–59. 10.3109/14767058.2015.1006620 PubMed DOI PMC

Chaemsaithong P, Romero R, Korzeniewski SJ, Martinez-Varea A, Dong Z, Yoon BH, et al. A point of care test for interleukin-6 in amniotic fluid in preterm prelabor rupture of membranes: a step toward the early treatment of acute intra-amniotic inflammation/infection. J Matern Fetal Neonatal Med. 2016;29(3):360–7. 10.3109/14767058.2015.1006621 PubMed DOI PMC

Papile LA, Burstein J, Burstein R, Koffler H. Incidence and evolution of subependymal and intraventricular hemorrhage: a study of infants with birth weights less than 1,500 gm. J Pediatr. 1978;92(4):529–34. Epub 1978/04/01. 10.1016/s0022-3476(78)80282-0 . PubMed DOI

Schwebke JR, Muzny CA, Josey WE. Role of Gardnerella vaginalis in the pathogenesis of bacterial vaginosis: a conceptual model. J Infect Dis. 2014;210(3):338–43. 10.1093/infdis/jiu089 . PubMed DOI

Machado D, Castro J, Martinez-de-Oliveira J, Nogueira-Silva C, Cerca N. Prevalence of bacterial vaginosis in Portuguese pregnant women and vaginal colonization by Gardnerella vaginalis. PeerJ. 2017;5:e3750 Epub 2017/09/07. 10.7717/peerj.3750 PubMed DOI PMC

Pillay K, Nzimande S, Naicker M, Ramsuran V, Tinarwo P, Abbai N. Prevalence of Genotypes and Subtypes of Gardnerella vaginalis in South African Pregnant Women. Infect Dis Obstet Gynecol. 2020;2020:3176407 Epub 2020/07/23. 10.1155/2020/3176407 PubMed DOI PMC

Nelson DB, Hanlon A, Nachamkin I, Haggerty C, Mastrogiannis DS, Liu C, et al. Early pregnancy changes in bacterial vaginosis-associated bacteria and preterm delivery. Paediatr Perinat Epidemiol. 2014;28(2):88–96. Epub 2014/01/11. 10.1111/ppe.12106 PubMed DOI PMC

McGregor JA, French JI, Seo K. Premature rupture of membranes and bacterial vaginosis. Am J Obstet Gynecol. 1993;169(2 Pt 2):463–6. 10.1016/0002-9378(93)90342-g . PubMed DOI

Theis KR, Romero R, Motomura K, Galaz J, Winters AD, Pacora P, et al. Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes. J Perinat Med. 2020;48(2):115–31. 10.1515/jpm-2019-0398 PubMed DOI PMC

Kacerovsky M, Pliskova L, Bolehovska R, Skogstrand K, Hougaard DM, Tsiartas P, et al. The impact of the microbial load of genital mycoplasmas and gestational age on the intensity of intraamniotic inflammation. Am J Obstet Gynecol. 2012;206(4):342 e1–8. 10.1016/j.ajog.2012.01.004 . PubMed DOI

Kacerovsky M, Pliskova L, Bolehovska R, Musilova I, Hornychova H, Tambor V, et al. The microbial load with genital mycoplasmas correlates with the degree of histologic chorioamnionitis in preterm PROM. Am J Obstet Gynecol. 2011;205(3):213 e1–7. 10.1016/j.ajog.2011.04.028 . PubMed DOI

Musilova I, Andrys C, Drahosova M, Zednikova B, Hornychova H, Pliskova L, et al. Late preterm prelabor rupture of fetal membranes: fetal inflammatory response and neonatal outcome. Pediatr Res. 2018;83(3):630–7. 10.1038/pr.2017.300 . PubMed DOI

Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Hale LP, et al. Adherent biofilms in bacterial vaginosis. Obstet Gynecol. 2005;106(5 Pt 1):1013–23. 10.1097/01.AOG.0000183594.45524.d2 . PubMed DOI

Swidsinski A, Verstraelen H, Loening-Baucke V, Swidsinski S, Mendling W, Halwani Z. Presence of a polymicrobial endometrial biofilm in patients with bacterial vaginosis. PLoS One. 2013;8(1):e53997 10.1371/journal.pone.0053997 PubMed DOI PMC

Santiago GL, Deschaght P, El Aila N, Kiama TN, Verstraelen H, Jefferson KK, et al. Gardnerella vaginalis comprises three distinct genotypes of which only two produce sialidase. Am J Obstet Gynecol. 2011;204(5):450 e1–7. 10.1016/j.ajog.2010.12.061 . PubMed DOI

Patterson JL, Stull-Lane A, Girerd PH, Jefferson KK. Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes. Microbiology. 2010;156(Pt 2):392–9. 10.1099/mic.0.034280-0 PubMed DOI PMC

Castro J, Machado D, Cerca N. Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm. Pathog Dis. 2016;74(3). 10.1093/femspd/ftw007 . PubMed DOI

Reid G, Sobel JD. Bacterial adherence in the pathogenesis of urinary tract infection: a review. Rev Infect Dis. 1987;9(3):470–87. 10.1093/clinids/9.3.470 . PubMed DOI

Brown RG, Marchesi JR, Lee YS, Smith A, Lehne B, Kindinger LM, et al. Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin. BMC Med. 2018;16(1):9 10.1186/s12916-017-0999-x PubMed DOI PMC

Gilbert NM, Lewis WG, Lewis AL. Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis. PLoS One. 2013;8(3):e59539 10.1371/journal.pone.0059539 PubMed DOI PMC

Sierra LJ, Brown AG, Barila GO, Anton L, Barnum CE, Shetye SS, et al. Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice. PLoS One. 2018;13(1):e0191524 10.1371/journal.pone.0191524 PubMed DOI PMC

Kim JY, Park SC, Lee JK, Choi SJ, Hahm KS, Park Y. Novel antibacterial activity of beta(2)-microglobulin in human amniotic fluid. PloS one. 2012;7(11):e47642 10.1371/journal.pone.0047642 PubMed DOI PMC

Oka K, Hagio Y, Tetsuoh M, Kawano K, Hamada T, Kato T. The effect of transferrin and lysozyme on antibacterial activity of amniotic fluid. Biological research in pregnancy and perinatology. 1987;8(1 1ST Half):1–6. . PubMed

Pleckaityte M, Janulaitiene M, Lasickiene R, Zvirbliene A. Genetic and biochemical diversity of Gardnerella vaginalis strains isolated from women with bacterial vaginosis. FEMS Immunol Med Microbiol. 2012;65(1):69–77. 10.1111/j.1574-695X.2012.00940.x . PubMed DOI

Janulaitiene M, Gegzna V, Baranauskiene L, Bulavaite A, Simanavicius M, Pleckaityte M. Phenotypic characterization of Gardnerella vaginalis subgroups suggests differences in their virulence potential. PLoS One. 2018;13(7):e0200625 10.1371/journal.pone.0200625 PubMed DOI PMC

Janulaitiene M, Paliulyte V, Grinceviciene S, Zakareviciene J, Vladisauskiene A, Marcinkute A, et al. Prevalence and distribution of Gardnerella vaginalis subgroups in women with and without bacterial vaginosis. BMC Infect Dis. 2017;17(1):394 10.1186/s12879-017-2501-y PubMed DOI PMC

Ahmed A, Earl J, Retchless A, Hillier SL, Rabe LK, Cherpes TL, et al. Comparative genomic analyses of 17 clinical isolates of Gardnerella vaginalis provide evidence of multiple genetically isolated clades consistent with subspeciation into genovars. J Bacteriol. 2012;194(15):3922–37. 10.1128/JB.00056-12 PubMed DOI PMC

Balashov SV, Mordechai E, Adelson ME, Gygax SE. Identification, quantification and subtyping of Gardnerella vaginalis in noncultured clinical vaginal samples by quantitative PCR. J Med Microbiol. 2014;63(Pt 2):162–75. 10.1099/jmm.0.066407-0 . PubMed DOI

Paramel Jayaprakash T, Schellenberg JJ, Hill JE. Resolution and characterization of distinct cpn60-based subgroups of Gardnerella vaginalis in the vaginal microbiota. PLoS One. 2012;7(8):e43009 10.1371/journal.pone.0043009 PubMed DOI PMC

Schellenberg JJ, Paramel Jayaprakash T, Withana Gamage N, Patterson MH, Vaneechoutte M, Hill JE. Gardnerella vaginalis Subgroups Defined by cpn60 Sequencing and Sialidase Activity in Isolates from Canada, Belgium and Kenya. PLoS One. 2016;11(1):e0146510 10.1371/journal.pone.0146510 PubMed DOI PMC

Prevalence and Load of Cervical Ureaplasma Species With Respect to Intra-amniotic Complications in Women With Preterm Prelabor Rupture of Membranes Before 34 weeks

Clinical characteristics of colonization of the amniotic cavity in women with preterm prelabor rupture of membranes, a retrospective study