Identification of Cultivable Bacteria in Amniotic Fluid Using Cervicovaginal Fluid Protein Microarray in Preterm Premature Rupture of Membranes

  • Seung Mi Lee
  • Kyo Hoon ParkEmail author
  • Subeen Hong
  • Yu Mi Kim
  • Ye Hyon Park
  • Young Eun Lee
  • Se Jeong Jeon
Original Article


We aimed to identify cervicovaginal fluid (CVF) protein biomarkers of microbial invasion of the amniotic cavity (MIAC) in women with preterm premature rupture of membranes (PPROM), using an antibody microarray. This retrospective cohort study included 99 consecutive women with singleton pregnancies and PPROM (23–33 weeks) who underwent amniocentesis and who gave CVF samples. CVF proteomes from the MIAC (n = 20) versus non-MIAC groups (n = 20) were comparatively profiled by an antibody microarray using a nested case-control study design. The seven candidate biomarkers of interest were validated in the total cohort (n = 99) by enzyme-linked immunosorbent assays (ELISA). For comparison with candidate markers, amniotic fluid (AF) white blood cell (WBC) count was also measured. The primary outcome measure was MIAC (defined as positive AF culture). Thirty of the proteins studied exhibited significant intergroup differences. Measurements of the total cohort with ELISA confirmed a significant increase in the levels of CVF IL-8, lipocalin-2, MIP-1α, MMP-9, and TIMP-1 in women with MIAC, independent of gestational age at sampling. A combined, non-invasive model was developed by using a stepwise regression procedure, which included CVF IL-8 and CVF MMP-9 (area under the curve [AUC] = 0.763), and this AUC was comparable with the AUC of AF WBC. Using protein–antibody microarray technology, we found several novel, independent, non-invasive biomarkers to identify MIAC in women with PPROM: IL-8, lipocalin-2, MIP-1α, MMP-9, and TIMP-1. Furthermore, the combined non-invasive model (IL-8 and MMP-9) was a useful independent predictor for MIAC with good discriminatory power, similar to AF WBC count.


Antibody microarray Biomarker Cervicovaginal fluid Microbial invasion of the amniotic cavity Preterm premature rupture of membranes 


Funding Information

This study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI18C0063).

Compliance with Ethical Standards

At enrollment, patients with PPROM provided consent for the collection and use of CVF samples and for the use of their clinical information for research purposes. This retrospective cohort study was approved by the Ethics Committee at Seoul National University Bundang Hospital, Seongnamsi, Republic of Korea (IRB no. B-1105/128-102).

Conflict of Interest

The authors declare that there is no conflict of interest.

Supplementary material

43032_2020_143_MOESM1_ESM.docx (20 kb)
ESM 1 (DOCX 20.1 kb)
43032_2020_143_MOESM2_ESM.docx (24 kb)
Table S1 (DOCX 24 kb)


  1. 1.
    Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet. 2008;371:75–84.PubMedCrossRefGoogle Scholar
  2. 2.
    Bulletins-Obstetrics ACoP. ACOG Practice Bulletin No. 80: premature rupture of membranes. Clinical management guidelines for obstetrician-gynecologists. Obstet Gynecol. 2007;109:1007–19.CrossRefGoogle Scholar
  3. 3.
    Armstrong-Wells J, Donnelly M, Post MD, Manco-Johnson MJ, Winn VD, Sebire G. Inflammatory predictors of neurologic disability after preterm premature rupture of membranes. Am J Obstet Gynecol. 2015;212:e211–9.CrossRefGoogle Scholar
  4. 4.
    Kibel M, Asztalos E, Barrett J, et al. Outcomes of pregnancies complicated by preterm premature rupture of membranes between 20 and 24 weeks of gestation. Obstet Gynecol. 2016;128:313–20.PubMedCrossRefGoogle Scholar
  5. 5.
    Goncalves LF, Chaiworapongsa T, Romero R. Intrauterine infection and prematurity. Ment Retard Dev Disabil Res Rev. 2002;8:3–13.PubMedCrossRefGoogle Scholar
  6. 6.
    Ryu A, Park KH, Oh KJ, Lee SY, Jeong EH, Park JW. Predictive value of combined cervicovaginal cytokines and gestational age at sampling for intra-amniotic infection in preterm premature rupture of membranes. Acta Obstet Gynecol Scand. 2013;92:517–24.PubMedCrossRefGoogle Scholar
  7. 7.
    Jung EY, Park KH, Han BR, Cho SH, Ryu A. Measurement of interleukin 8 in cervicovaginal fluid in women with preterm premature rupture of membranes: a comparison of amniotic fluid samples. Reprod Sci. 2017;24:142–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Cobo T, Jacobsson B, Kacerovsky M, et al. Systemic and local inflammatory response in women with preterm prelabor rupture of membranes. PLoS One. 2014;9:e85277.PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Musilova I, Andrys C, Drahosova M, Soucek O, Pliskova L, Jacobsson B, et al. Cervical fluid interleukin 6 and intra-amniotic complications of preterm prelabor rupture of membranes. J Matern Fetal Neonatal Med. 2018;31:827–36.PubMedCrossRefGoogle Scholar
  10. 10.
    Kacerovsky M, Musilova I, Jacobsson B, et al. Cervical fluid IL-6 and IL-8 levels in pregnancies complicated by preterm prelabor rupture of membranes. J Matern Fetal Neonatal Med. 2015;28:134–40.PubMedCrossRefGoogle Scholar
  11. 11.
    Jun JK, Yoon BH, Romero R, Kim M, Moon JB, Ki SH, et al. Interleukin 6 determinations in cervical fluid have diagnostic and prognostic value in preterm premature rupture of membranes. Am J Obstet Gynecol. 2000;183:868–73.PubMedCrossRefGoogle Scholar
  12. 12.
    Lee SM, Park KH, Jung EY, Kook SY, Park H, Jeon SJ. Inflammatory proteins in maternal plasma, cervicovaginal and amniotic fluids as predictors of intra-amniotic infection in preterm premature rupture of membranes. PLoS One. 2018;13:e0200311.PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Price JV, Haddon DJ, Kemmer D, et al. Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus. J Clin Invest. 2013;123:5135–45.PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Sutandy FX, Qian J, Chen CS, Zhu H. Overview of protein microarrays. Curr Protoc Protein Sci. 2013;Chapter 27:Unit 27.Google Scholar
  15. 15.
    Duarte JG, Blackburn JM. Advances in the development of human protein microarrays. Expert Rev Proteomics. 2017;14:627–41.PubMedCrossRefGoogle Scholar
  16. 16.
    Lee SY, Park KH, Jeong EH, Oh KJ, Ryu A, Kim A. Intra-amniotic infection/inflammation as a risk factor for subsequent ruptured membranes after clinically indicated amniocentesis in preterm labor. J Korean Med Sci. 2013;28:1226–32.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Park KH, Kim SN, Oh KJ, Lee SY, Jeong EH, Ryu A. Noninvasive prediction of intra-amniotic infection and/or inflammation in preterm premature rupture of membranes. Reprod Sci. 2012;19:658–65.PubMedCrossRefGoogle Scholar
  18. 18.
    Park JW, Park KH, Lee JE, Kim YM, Lee SJ, Cheon DH. Antibody microarray analysis of plasma proteins for the prediction of histologic chorioamnionitis in women with preterm premature rupture of membranes. Reprod Sci 2019:1933719119828043.Google Scholar
  19. 19.
    Cha DM, Woo SJ, Kim HJ, Lee C, Park KH. Comparative analysis of aqueous humor cytokine levels between patients with exudative age-related macular degeneration and normal controls. Invest Ophthalmol Vis Sci. 2013;54:7038–44.PubMedCrossRefGoogle Scholar
  20. 20.
    DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics. 1988;44:837–45.CrossRefGoogle Scholar
  21. 21.
    Cobo T, Kacerovsky M, Holst RM, et al. Intra-amniotic inflammation predicts microbial invasion of the amniotic cavity but not spontaneous preterm delivery in preterm prelabor membrane rupture. Acta Obstet Gynecol Scand. 2012;91:930–5.PubMedCrossRefGoogle Scholar
  22. 22.
    Park JW, Park KH, Jung EY. Clinical significance of histologic chorioamnionitis with a negative amniotic fluid culture in patients with preterm labor and premature membrane rupture. PLoS One. 2017;12:e0173312.PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Oh KJ, Park KH, Kim SN, Jeong EH, Lee SY, Yoon HY. Predictive value of intra-amniotic and serum markers for inflammatory lesions of preterm placenta. Placenta. 2011;32:732–6.PubMedCrossRefGoogle Scholar
  24. 24.
    Cobo T, Tsiartas P, Kacerovsky M, Holst RM, Hougaard DM, Skogstrand K, et al. Maternal inflammatory response to microbial invasion of the amniotic cavity: analyses of multiple proteins in the maternal serum. Acta Obstet Gynecol Scand. 2013;92:61–8.PubMedCrossRefGoogle Scholar
  25. 25.
    Locksmith GJ, Clark P, Duff P, Saade GR, Schultz GS. Amniotic fluid concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during pregnancy and labor. Am J Obstet Gynecol. 2001;184:159–64.PubMedCrossRefGoogle Scholar
  26. 26.
    Zariffard MR, Anastos K, French AL, et al. Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract. PLoS One. 2015;10:e0116911.PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Devarajan P. Review: neutrophil gelatinase-associated lipocalin: a troponin-like biomarker for human acute kidney injury. Nephrology (Carlton). 2010;15:419–28.CrossRefGoogle Scholar
  28. 28.
    Nasioudis D, Witkin SS. Neutrophil gelatinase-associated lipocalin and innate immune responses to bacterial infections. Med Microbiol Immunol. 2015;204:471–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Beghini J, Giraldo PC, Linhares IM, Ledger WJ, Witkin SS. Neutrophil gelatinase-associated lipocalin concentration in vaginal fluid: relation to bacterial vaginosis and vulvovaginal candidiasis. Reprod Sci. 2015;22:964–8.PubMedCrossRefGoogle Scholar
  30. 30.
    Rood KM, Buhimschi IA, Rodewald Millen K, et al. Evidence for participation of neutrophil gelatinase-associated lipocalin/matrix metalloproteinase-9 (NGAL*MMP-9) complex in the inflammatory response to infection in pregnancies complicated by preterm birth. Am J Reprod Immunol. 2016;76:108–17.PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Wolpe SD, Davatelis G, Sherry B, et al. Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med. 1988;167:570–81.PubMedCrossRefGoogle Scholar
  32. 32.
    Romero R, Gomez R, Galasso M, et al. Macrophage inflammatory protein-1 alpha in term and preterm parturition: effect of microbial invasion of the amniotic cavity. Am J Reprod Immunol. 1994;32:108–13.PubMedCrossRefGoogle Scholar
  33. 33.
    Wang T, Lv JH, Zhang XF, Li CJ, Han X, Sun YJ. Tissue inhibitor of metalloproteinase-1 protects MCF-7 breast cancer cells from paclitaxel-induced apoptosis by decreasing the stability of cyclin B1. Int J Cancer. 2010;126:362–70.PubMedCrossRefGoogle Scholar
  34. 34.
    Stetler-Stevenson WG. Tissue inhibitors of metalloproteinases in cell signaling: metalloproteinase-independent biological activities. Sci Signal. 2008;1:re6.PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Sundrani D, Narang A, Mehendale S, Joshi S, Chavan-Gautam P. Investigating the expression of MMPs and TIMPs in preterm placenta and role of CpG methylation in regulating MMP-9 expression. IUBMB Life. 2017;69:985–93.PubMedCrossRefGoogle Scholar
  36. 36.
    Athayde N, Romero R, Gomez R, et al. Matrix metalloproteinases-9 in preterm and term human parturition. J Matern Fetal Med. 1999;8:213–9.PubMedGoogle Scholar
  37. 37.
    Lee SE, Romero R, Lee SM, Yoon BH. Amniotic fluid volume in intra-amniotic inflammation with and without culture-proven amniotic fluid infection in preterm premature rupture of membranes. J Perinat Med. 2010;38:39–44.PubMedPubMedCentralGoogle Scholar
  38. 38.
    Lee J, Romero R, Kim SM, Chaemsaithong P, Yoon BH. A new antibiotic regimen treats and prevents intra-amniotic inflammation/infection in patients with preterm PROM. J Matern Fetal Neonatal Med. 2016;29:2727–37.PubMedGoogle Scholar
  39. 39.
    Committee on Practice B-O. ACOG Practice Bulletin No. 188: prelabor rupture of membranes. Obstet Gynecol. 2018;131:e1–e14.CrossRefGoogle Scholar
  40. 40.
    Redelinghuys MJ, Ehlers MM, Dreyer AW, Lombaard HA, Kock MM. Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women. BMC Infect Dis. 2014;14:171.PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Combs CA, Gravett M, Garite TJ, et al. Amniotic fluid infection, inflammation, and colonization in preterm labor with intact membranes. Am J Obstet Gynecol. 2014;210:e121–5.CrossRefGoogle Scholar
  42. 42.
    Ferreira CST, da Silva MG, de Pontes LG, Dos Santos LD, Marconi C. Protein content of cervicovaginal fluid is altered during bacterial vaginosis. J Low Genit Tract Dis. 2018;22:147–51.PubMedCrossRefGoogle Scholar
  43. 43.
    Noda-Nicolau NM, Bastos LB, Bolpetti AN, Pinto GVS, Marcolino LD, Marconi C, et al. Cervicovaginal levels of human beta-defensin 1, 2, 3, and 4 of reproductive-aged women with chlamydia trachomatis infection. J Low Genit Tract Dis. 2017;21:189–92.PubMedCrossRefGoogle Scholar
  44. 44.
    Witkin SS, Moron AF, Ridenhour BJ, et al. Vaginal biomarkers that predict cervical length and dominant bacteria in the vaginal microbiomes of pregnant women. MBio. 2019;10:e02242–19.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Society for Reproductive Investigation 2020

Authors and Affiliations

  • Seung Mi Lee
    • 1
    • 2
  • Kyo Hoon Park
    • 1
    • 3
    Email author
  • Subeen Hong
    • 1
    • 3
  • Yu Mi Kim
    • 3
  • Ye Hyon Park
    • 3
  • Young Eun Lee
    • 3
  • Se Jeong Jeon
    • 1
  1. 1.Department of Obstetrics and GynecologySeoul National University College of MedicineSeoulSouth Korea
  2. 2.Department of Obstetrics and GynecologySeoul National University HospitalSeoulSouth Korea
  3. 3.Department of Obstetrics and GynecologySeoul National University Bundang HospitalSeongnamsiSouth Korea

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