Ureaplasma urealyticum is the microorganism most frequently isolated from the amniotic fluid of women in preterm labor. The relationship between intra-amniotic U. urealyticum in healthy second-trimester pregnant women and subsequent pregnancy outcome was investigated. Transabdominal amniotic fluid obtained from 254 asymptomatic women at 15-17 weeks' gestation were tested by polymerase chain reaction (PCR). U. urealyticum was identified in 29 subjects (11.4%). A subsequent preterm labor occurred in 17 U. urealyticum-positive women (58.6%), compared with 10 (4.4%) U. urealyticum-negative women (P<.0001). Preterm birth was documented in 7 (24.1%) U. urealyticum-positive women compared with only 1 U. urealyticum-negative woman (0.4%) (P<.0001). U. urealyticum-positive women also had a higher prevalence of preterm labor in a prior pregnancy (20.7%) than did the negative women (2.7%; P=.0008). PCR testing of second-trimester amniotic fluid for U. urealyticum can identify women at risk for subsequent preterm labor and deliver

Gerber, Stefan

Hohlfeld, Patrick

Vial, Yvan

Witkin, Steven S.

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518 • JID 2003:187 (1 February) • BRIEF REPORTB R I E F R E P O R TDetection of Ureaplasma urealyticumin Second-Trimester Amniotic Fluidby Polymerase Chain ReactionCorrelates with Subsequent PretermLabor and DeliveryStefan Gerber,1,2 Yvan Vial,2 Patrick Hohlfeld,2 and Steven S. Witkin11Division of Immunology and Infectious Diseases, Department of Obstetricsand Gynecology, Weill Medical College of Cornell University, New York, NewYork; 2Department of Gynecology and Obstetrics, Centre HospitalierUniversitaire Vandois, Lausanne, SwitzerlandUreaplasma urealyticum is the microorganism most frequent-ly isolated from the amniotic fluid of women in preterm la-bor. The relationship between intra-amniotic U. urealyticum inhealthy second-trimester pregnant women and subsequentpreg-nancy outcome was investigated. Transabdominal amnioticfluidobtained from 254 asymptomatic women at 15–17 weeks’ ges-tation were tested by polymerase chain reaction (PCR). U. urea-lyticum was identified in 29 subjects (11.4%). A subsequentpreterm labor occurred in 17 U. urealyticum–positive women(58.6%), compared with 10 (4.4%) U. urealyticum–negativewomen ( ). Preterm birth was documented in 7 (24.1%)P ! .0001U. urealyticum–positive women compared with only 1 U. urea-lyticum–negative woman (0.4%) ( ). U. urealyticum–P ! .0001positive women also had a higher prevalence of preterm laborin a prior pregnancy (20.7%) than did the negative women(2.7%; ). PCR testing of second-trimester amnioticPp .0008fluid for U. urealyticum can identify women at risk for subse-quent preterm labor and delivery.Ureaplasma urealyticum frequently colonizes the lower geni-tal tract of pregnant women, without apparent adverse con-Received 9 August 2002; revised 17 October 2002; electronically published 24 January 2003.Presented in part: 7th annual meeting of the International Infectious Diseases Society for Obstetricsand Gynecology, Pasadena, California, May 2002.Financial support: National Institutes of Health (grant HD41676).Informed consent was obtained from patients, and human experimentation guidelines of the USDepartment of Health and Human Services and Centre Hospitalier, Lausanne, Switzerland, werefollowed in the conduct of clinical research.Reprints or correspondence: Dr. Steven S. Witkin, Dept. of Obstetrics and Gynecology, Weill MedicalCollege of Cornell University, 525 E. 68th St., Box 35, New York, NY 10021 (switkin@med.cornell.edu).The Journal of Infectious Diseases 2003; 187:518–21 2003 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2003/18703-0023$15.00sequences [1]. In a small subpopulation of women, this mi-croorganism ascends and colonizes the endometrium, eitherbefore or after conception [2]. The difficulty resides in iden-tifying the group at risk for ascending colonization. Passage ofU. urealyticum into the amniotic cavity appears to be an im-portant step for prognosis of the pregnancy. Colonization ofthe amniotic fluid has been documented as a major risk factor,but usually the diagnosis is made too late for successful inter-vention [3]. To minimize a possible adverse influence of U.urealyticum in pregnancy outcome, a protocol to successfullyidentify this microorganism early in the pregnancy would bebeneficial.U. urealyticum is the microorganism most frequently cul-tured by use of standard culture techniques from amnioticfluids of women in preterm labor (PTL) with intact membranes[4] or with preterm premature rupture of membranes (P-PROM) [5]. Whether U. urealyticum intra-amniotic coloni-zation in asymptomatic women during the second trimester isalso a risk factor for subsequent adverse pregnancy outcomehas been examined in only a few studies. U. urealyticum cul-ture–positive amniotic fluids have been observed in a smallpercentage of healthy asymptomatic women in the second tri-mester of pregnancy [6–9]. In each of these studies, a positiveU. urealyticum culture was associated with an increased risk ofadverse pregnancy outcome in untreated patients, comparedwith those who were culture-negative or who received antibiotictreatment for this microorganism.A recent study that used polymerase chain reaction (PCR)to detect U. urealyticum in amniotic fluids from women withP-PROM demonstrated that PCR was more sensitive than cul-ture in detecting this microorganism in amniotic fluid and thatPCR-positive, culture-negative women were at high risk foradverse pregnancy outcomes [10]. The objective of the presentinvestigation was to perform PCR testing on a large numberof second-trimester amniotic fluids from healthy asymptomaticwomen to evaluate the prevalence of this mycoplasma andwhether its detection is associated with P-PROM or pretermbirth.Subjects, materials, and methods. Amniotic fluid sampleswere collected for analysis from 317 consecutive women whounderwent a transabdominal amniocentesis at weeks 15–17 ofpregnancy and were stored at 80C. Indications for amnio-centesis were advanced maternal age, family history of chro-mosomal abnormality, maternal request, or positive detectionon “triple test” for alpha fetoprotein, estriol, and beta humanchorionic gonadotropin. The same fetal ultrasound team per-BRIEF REPORT • JID 2003:187 (1 February) • 519Table 1. Relationship between Ureaplasma urealyticum in amni-otic fluid and historical variables.VariableU. urealyticum statusPPositive NegativePregnancy, mean no. (range) 2.3 (1–5) 2.5 (1–9)Birth, mean no. (range) 0.9 (0–4) 1.0 (0–4)Age, mean years (range) 34.5 (25–42) 34.0 (19–42)Previous preterm birth, no. (%) 6 (20.7) 6 (2.7) .0008Table 2. Relationship between Ureaplasma urealyticum in amni-otic fluid and pregnancy outcome.Outcome variableU. urealyticum statusPPositive(np 29)Negative(np 225)PTL 17 (58.6) 10 (4.4) !.0001Hospital stay for PTL 9 (31.0) 6 (2.7) !.0001BirthBefore 37 weeks 7 (24.1) 1 (0.4) !.0001Before 34 weeks 2 (6.9) 0 .01P-PROM 6 (20.7) 1 (0.4) !.0001Cesarean section 9 (31.0) 56 (24.9) NSNOTE. Data are no. (%) of subjects. NS, not significant; P-PROM, pretermpremature rupture of membranes; PTL, preterm labor.formed all procedures, using the identical method in every case.Before the transabdominal amniocentesis, the skin was disin-fected with 10% povidine iodine.All women were white and of European background. Sub-sequent pregnancy follow-up revealed that 63 (19.9%) of thesubjects had either an elective pregnancy termination or a com-plicating pregnancy factor that placed them at risk for an ad-verse outcome: placenta praevia, intrauterine growth restric-tion, hypertension, cervical incompetence, or twin gestation.The remaining 254 women had no known risk factor for ad-verse pregnancy outcome.U. urealyticum in amniotic fluid was detected by PCR usinga procedure detailed elsewhere [11], in which primer pairs toa region of the urease gene are used, except that digoxigenin-labeled dUTP was added to the reaction mixture. To ensurespecificity, the PCR amplicons were hybridized to a biotinylat-ed oligonucleotide internal probe (5′-biotin-GCC CAC CAAGAC TAT GAT GTT TAG-3′) and the complex detected induplicate by ELISA using streptavidin-coated wells of a micro-titer plate and peroxidase-labeled anti-digoxigenin antibody(Roche Diagnostics).Stringent precautions used to prevent PCR product carryoverwere the frequent changing of gloves and performance of theprocessing, PCR, and hybridization in different rooms. Sampleswere analyzed without knowledge of pregnancy outcomes.Clinical pregnancy outcome data were obtained only afterthe completion of all testing in a blinded fashion directly fromthe patient and her private obstetrician. “PTL” was defined asregular uterine contractions that indicated the use of a tocolyticdrug and bed rest at home after the exclusion of other etiologies(i.e., bladder infection, cervical infection, or cervical incom-petence). P-PROM was diagnosed by objective amniotic fluidleakage (based on history, physical examination, and laboratorytesting) and/or diminution of amniotic fluid index before 37weeks of pregnancy. In such cases, the condition of the patientwas managed by observation, in the absence of any infectionor fetal distress.The relation between PCR outcome and other variables wasanalyzed by Fisher’s exact test. was considered to beP ! .05significant.Results. During the 6-month study period, the same groupof physicians performed 317 amniocenteses. Of these, there was1 pregnancy loss 2 weeks after, and 2 other patients developedvaginal bleeding 24 and 48 h after the amniocentesis. Bothrecovered after prolonged bed rest. Subsequent examination ofmedical records revealed that 16 patients had an elective ter-mination of the pregnancy, 2 had cervical incompetence, and45 had a major complicating pregnancy factor. All were negativefor U. urealyticum, except for 2 sets of twin children.U. urealyticum was detected by PCR in amniotic fluid from29 (11.4%) of the remaining 254 asymptomatic women witha singleton pregnancy. The relationship between U. urealyticumdetection and historical variables in these patients is shown intable 1. There was no relationship between U. urealyticum col-onization and the age or number of previous pregnancies orbirths. However, women positive for U. urealyticum had ahigher occurrence of PTL in a prior pregnancy (20.7%) thandid the PCR-negative women (2.7%; ). Eight of thePp .0008amniotic fluid samples were visibly discolored; none of thesewere positive for U. urealyticum.The relationship between U. urealyticum detection and out-come of the pregnancy is shown in table 2. Intra-amnioticcarriage of U. urealyticum was highly associated with PTL, hos-pitalization for PTL, P-PROM, and preterm birth before 37weeks ( ). Delivery before 34 weeks’ gestation was ob-P ! .0001served in 2 of the U. urealyticum–positive women and in noneof the U. urealyticum–negative women ( ).Pp .01Discussion. In our study of 254 asymptomatic pregnantpatients in their early second trimester, 3% subsequently de-veloped P-PROM and 10% underwent PTL, rates that were inagreement with other studies [12]. Similarly, our incidence ofpregnancy loss (0.3%) and complication after midtrimester am-niocentesis (0.9%) corresponded to traditional rates [13].In the present study, detection of U. urealyticum by PCR insecond-trimester amniotic fluid of asymptomatic women washighly correlated with subsequent PTL and preterm delivery.The findings are similar to earlier investigations that examinedthe impact on pregnancy of having a positive second-trimester520 • JID 2003:187 (1 February) • BRIEF REPORTamniotic fluid culture for U. urealyticum [6–9]. However, thepercentage of U. urealyticum–positive amniotic fluid samplesin our study exceeded that found in previous investigationsthat used culture techniques. Possible explanations include theenhanced sensitivity of PCR over culture for detection of thismicroorganism in amniotic fluid [10] and/or population dif-ferences in lower genital tract colonization or migration of U.urealyticum to the upper genital tract. The extent of U. urealy-ticum colonization is known to vary widely among populationsin different geographic areas [8]. U. urealyticum cultures werenot performed on amniotic fluids in the present study, so weare unable to compare the sensitivity of our PCR with culturefor these samples.Women in the present study who were positive for intra-amniotic U. urealyticum also had a higher rate of PTL in pre-vious pregnancies than did PCR-negative women. This obser-vation is consistent with the possibility that U. urealyticummight have been present in the endometrium as a persistentcolonizer before the current conception. This raises the inter-esting possibility that it might be of value to sample the en-dometrium for U. urealyticum prior to attempted conceptionin women with a prior preterm birth. Detection and treatmentmight improve subsequent pregnancy outcome. It has beenproposed elsewhere that preexisting infection of the uterinecavity was a predisposing factor for subsequent adverse preg-nancy outcomes [14].It remains uncertain whether U. urealyticum directly inducedthe observed pregnancy complications or whether this organ-ism was merely a marker for another infection. It has beensuggested that the significance of U. urealyticum in amnioticfluid is mainly its association with other microorganisms [15].However, when standard culture techniques are used, U. urea-lyticum is often the sole microorganism identified in the am-niotic fluid of women in PTL and, furthermore, is associatedwith elevated intra-amniotic levels of proinflammatory cyto-kines [4, 6, 9, 10]. Studies that use more refined gene ampli-fication analyses are necessary to definitively rule out the pres-ence of additional microorganisms in U. urealyticum–positiveamniotic fluid samples and their relationship to adverse preg-nancy outcome.It should be noted that 76% of our patients with intra-amnioticU. urealyticum gave birth at 37 weeks, whereas only 21% ofthe U. urealyticum–positive patients developed P-PROM and24% gave birth preterm. It would be of great interest to identifymarkers predictive of adverse pregnancy outcomes in the U.urealyticum–positive group. Perhaps a quantitative PCR or acombination of PCR and culture to identify the extent of infec-tion, would be of value. Evaluation of the inflammatory responsein amniotic fluid, determined by levels of proinflammatory cy-tokines and matrix metalloproteinases, or the extent of a stressresponse, measurable by heat shock protein determination, couldprovide details of the fetal response to this microorganism. Inrecent unpublished studies from our laboratory, possession ofallele 2 of the polymorphic interleukin-1 receptor antagonist genealso appeared to increase the rate of adverse pregnancy outcomeafter U. urealyticum infection. A detailed evaluation of a com-bination of such markers may eventually lead to a method todefine the high-risk group in whom antibiotic treatment mayprove beneficial.Nevertheless, the highly significant association between intra-amniotic U. urealyticum and adverse pregnancy outcomestrongly suggests the potential value of PCR testing of pregnantwomen in their second trimester for this organism. Routinescreening for U. urealyticum in amniotic fluid at the time ofmidtrimester amniocentesis can identify the group of patientswho are infected with this microorganism. Further studies areneeded to assess whether all women with risk factors for pre-maturity, or all women who undergo a second-trimester am-niocentesis for other indications, might benefit from U. urea-lyticum amniotic fluid testing. In addition, the possible use ofnew antibiotic treatments such as azithromycin, which ex-hibits an increased ease of transplacental passage, to improvepregnancy outcome in U. urealyticum intra-amniotic infectionawaits further investigation.References1. Cassell GH, Waites KB, Watson HL, Crouse DT, Harasawa R. Urea-plasma urealyticum intrauterine infection: role in prematurity and dis-ease in newborns. Clin Microbiol Rev 1993; 6:69–87.2. Cassell GH, Younger BJ, Blackwell RE, et al. Microbiologic study ofinfertile women at the time of diagnostic laparoscopy: association ofUreaplasma urealyticum with a defined subpopulation. N Engl J Med1983; 308:502–6.3. Wang EE, Matlow AG, Ohlsson A, Nelson SC. Ureaplasma urealyticuminfections in the perinatal period. Clin Perinatol 1997; 24:91–105.4. Yoon BH, Chang JW, Romero R. Isolation of Ureaplasma urealyticumfrom the amniotic cavity and adverse outcome in preterm labor. ObstetGynecol 1998; 92:77–82.5. Yoon BH, Romero R, Park JS, et al. Microbial invasion of the amnioticcavity with Ureaplasma urealyticum is associated with a robust hostresponse in fetal, amniotic, and maternal compartments. Am J ObstetGynecol 1998; 179:1254–60.6. Cassell GH, Davis RO, Waites KB, et al. Isolation of Mycoplasma hominisand Ureaplasma urealyticum from amniotic fluid at 16–20 weeks of ges-tation: potential effect on outcome of pregnancy. Sex Transm Dis 1983;10(Suppl 4):294–302.7. Gray DF, Robinson HB, Malone J. Adverse outcome in pregnancyfollowing amniotic fluid isolation of Ureaplasma urealyticum. PrenatDiagn 1992; 12:111–7.8. Horowitz S, Horowitz J, Mazor M, Porath A, Glezerman M. Ureaplasmaurealyticum cervical colonization as a marker for pregnancy compli-cations. Int J Gynecol Obstet 1995; 48:15–9.9. Bashiri A, Horowitz S, Huleihel M, Hackmon R, Dukler D, Mazor M.Elevated concentrations of interleukin-6 in intra-amniotic infectionwith Ureaplasma urealyticum in asymptomatic women during geneticamniocentesis. Acta Obstet Gynecol Scand 1999; 78:379–82.10. Yoon BH, Romero R, Kim M, et al. Clinical implications of detectionof Ureaplasma urealyticum in the amniotic cavity with the polymerasechain reaction. Am J Obstet Gynecol 2000; 183:1130–7.BRIEF REPORT • JID 2003:187 (1 February) • 52111. Blanchard A, Hentschel J, Duffy L, Baldus K, Cassell GH. Detectionof Ureaplasma urealyticum by polymerase chain reaction in the uro-genital tract of adults, in amniotic fluid, and in the respiratory tractof newborns. Clin Infect Dis 1993; 17(Suppl 1):S148–53.12. American College of Obstetricians and Gynecologists (ACOG). Pretermlabor in precis: obstetrics. Washington, DC: ACOG, 1999:50–3.13. Blackwell SC, Abundis MG, Nehra PC. Five-year experience with mid-trimester amniocentesis performed by a single group of obstetricians-gynecologists at a community hospital. Am J Obstet Gynecol 2002;186:1130–2.14. Toth M, Witkin SS, Ledger WJ, Thaler H. The role of infection in theetiology of preterm birth. 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Detection of Ureaplasma urealyticum in Second-Trimester Amniotic Fluid by Polymerase Chain Reaction Correlates with Subsequent Preterm Labor and Delivery

Detection of Ureaplasma urealyticum in Second-Trimester Amniotic Fluid by Polymerase Chain Reaction Correlates with Subsequent Preterm Labor and Delivery

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