- Email: [email protected]
Microbial invasion of the amniotic cavity in midtrimester pregnancies using molecular microbiology
Accepted Manuscript Microbial Invasion of the Amniotic Cavity in Mid-Trimester Pregnancies Using Molecular Microbiology Shelley Rowlands, MD, Jennifer A. Danielewski, PhD, Sepehr N. Tabrizi, PhD, Susan P. Walker, MD, Suzanne M. Garland, MD PII:
S0002-9378(17)30383-6
DOI:
10.1016/j.ajog.2017.02.051
Reference:
YMOB 11571
To appear in:
American Journal of Obstetrics and Gynecology
Received Date: 21 February 2017 Revised Date:
26 February 2017
Accepted Date: 27 February 2017
Please cite this article as: Rowlands S, Danielewski JA, Tabrizi SN, Walker SP, Garland SM, Microbial Invasion of the Amniotic Cavity in Mid-Trimester Pregnancies Using Molecular Microbiology, American Journal of Obstetrics and Gynecology (2017), doi: 10.1016/j.ajog.2017.02.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Microbial invasion of the amniotic cavity in mid-trimester pregnancies using molecular microbiology.
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Shelley ROWLANDS1,2, MD; Jennifer A. DANIELEWSKI3, PhD; Sepehr N. TABRIZI2,3, PhD; Susan
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P. WALKER4,5, MD; Suzanne M. GARLAND2,3, MD
Department of Maternity Services, Epworth Freemasons Hospital, East Melbourne, 3002
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Victoria, Australia.
Department of Obstetrics and Gynaecology, University of Melbourne,
Royal Women’s Hospital, Parkville, 3052 Victoria, Australia.
Department of Microbiology and Infectious Diseases, Murdoch Childrens Research
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Institute, Parkville, 3052, Victoria, Australia.
Department of Perinatal Medicine, Mercy Hospital for Women, Heidelberg, 3084, Victoria,
Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for
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Women, Heidelberg, 3084, Victoria, Australia
Word Count: Abstract 209, Main text 2741
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ACCEPTED MANUSCRIPT Funding source: This study was supported in part by the Royal Women's Hospital Education fund and the
Conflict of Interest / Disclosure statement
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The authors report no conflict of interest.
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Microbiology Infectious Diseases Research fund.
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Corresponding Author: Professor Suzanne Garland, Department of Microbiology and Infectious Diseases, The Royal Women's Hospital, Locked Bag 300, Parkville, 3052, Victoria, Australia.
Fax: + 61-3-9347 8235
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Phone: + 61-3-8345 3670
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Condensation Subclinical colonization of the amniotic cavity with microorganisms is rare in the mid trimester among low risk women. In 344 women between 15 and 22 weeks of gestation no
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microbial invasion of the amniotic cavity was detected using species specific PCRs, as well as 16s and 18s approaches.
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Short version of title
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Microorganisms were not detected in the amniotic fluid in mid-trimester of pregnancy
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ACCEPTED MANUSCRIPT ABSTRACT Objective To determine the frequency of microbial invasion of the amniotic cavity in the midtrimester
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of pregnancy in patients undergoing amniocentesis for clinical indications. Study Design
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Prospective investigation of the amniotic fluid of 344 asymptomatic women recruited in mid pregnancy for the presence of microbial DNA. Amniotic samples obtained at time of
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amniocentesis for genetic testing on women between 15-22 weeks gestation were tested specifically for the presence of Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium, as well as for other bacteria and fungi using broadrange PCR only. Pregnancy outcomes were reviewed independent of all molecular test
Results
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results.
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Using broad-range PCR, the prevalence of microbial invasion of the amniotic cavity in women between 15 and 22 weeks of gestation was 0% (0 vs. 344). Early preterm delivery
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occurred in only 4 (1%) women; 1 delivered electively and 3 spontaneously. None were associated with U. urealyticum, U.parvum, M.hominis, or M. genitalium. In addition, broad range PCR did not reveal the presence of other bacterial or fungal microbes. Conclusions Microbial invasion of the amniotic cavity in midtrimester gestations of low-risk pregnant women was not detected using molecular methods in 344 patients.
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ACCEPTED MANUSCRIPT Keywords Preterm birth, mid-trimester, amniotic fluid, intraamniotic infection, chorioamnionitis,
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sterile biological fluid, molecular microbiology, fungi, mycoplasma, ureaplasma.
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ACCEPTED MANUSCRIPT INTRODUCTION Studies have investigated the presence of microorganisms in amniotic fluid among asymptomatic women from amniocentesis performed in the mid-trimester for genetic screening purposes. Some,1-3 but not all4 studies have suggested that U. urealyticum or M.
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hominis in the amniotic fluid may be associated with an increased risk for preterm pre-labor rupture of membranes (PPROM), preterm labor (PTL) and/or delivery (PTD). The role of
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Mycoplasma species and their clinical significance in the amniotic fluid of asymptomatic women remains unresolved. Further studies are needed to understand the aetiology and
amniotic microbial invasion.
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pathogenesis of preterm labor in relation to the presence or absence of asymptomatic intra-
We conducted a longitudinal study on a low risk cohort of healthy asymptomatic women, to
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determine the prevalence of intra- amniotic microbial invasion and to examine their subsequent pregnancy outcomes. Amniotic fluid sampled between 15 and 22 weeks
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gestation was assessed for the presence of microorganisms in the amniotic cavity, by species specific PCR for U. urealyticum, U. parvum, M. hominis and M. genitalium, and
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presence of other bacterial and fungal pathogens using broad-range 16S and 18S PCR, respectively.
MATERIALS AND METHODS Patient population Women were recruited from two major tertiary referral centres in Melbourne, Australia: the
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ACCEPTED MANUSCRIPT Royal Women’s Hospital, and The Mercy Hospital for Women, between September 2004 and December 2008. Study inclusion criteria were women undergoing amniocentesis for genetic prenatal diagnosis between 15-22 weeks gestation in a singleton pregnancy. Women were excluded if a fetal abnormality were identified on ultrasound or if they had
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current clinical evidence of infection (of any type) for which they had received antibiotic treatment within the preceding two weeks. Consecutive women were approached and the
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majority agreed to participate in the study. Gestation was established by ultrasound scan between 8-14 weeks. All women provided written informed consent. Ethics approval was
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received from the Research and Ethics Committees at both hospitals.
Sample processing
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Amniotic fluid collected at the time of amniocentesis was stored at -80oC until all samples were collected and then processed over the following one to two years. Processing involved two different preparation methods prior to DNA isolation: (i) concentration of amniotic
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fluid whereby one ml was centrifuged at 14,000x g for 15 minutes to ensure all biological particles were captured, with the resultant pellet resuspended in 200 µl of phosphate
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buffered saline; and (ii) 200 µl of neat (undiluted, non-centrifuged) amniotic fluid. The automated MagNA Pure LC extraction system (Roche Diagnostics, Mannheim, Germany) was used to isolate DNA with the MagNA Pure LC DNA Isolation Kit I on amniotic fluid preparations (i) and (ii). To improve the extraction efficiency, 33.3µg/ml of poly(A) RNA carrier was added to the lysis buffer, due to the potential for low concentrations of bacterial and fungal DNA in these samples. Nucleic acid was eluted into a final volume of 100 µl. To
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ACCEPTED MANUSCRIPT determine cell adequacy, and as an internal control for extraction and amplification procedures, qPCR targeting a 260 bp region of the human ß-globin gene was performed.5,6
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Clinical definitions and outcomes Microbial invasion of the amniotic cavity was defined as the detection of any bacterial or fungal microorganism in the amniotic fluid using the species specific and broad range 16S
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and 18S assays outlined below. In assessing subsequent pregnancy outcomes; early
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preterm birth was defined as birth occurring prior to 34 completed weeks’ gestation, preterm labour was defined as regular uterine contractions and cervical change at <34 completed weeks gestation, where intervention such as tocolysis, cervical cerclage or administration of prophylactic steroids for fetal lung maturation were considered to be
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indicated and PPROM was defined as spontaneous rupture of membranes prior to the onset of labor and before 34 completed weeks’ gestation. Medical record reviews were used to determine delivery outcomes for women who delivered at the recruiting hospitals, while
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outcomes for women who delivered at a non-recruiting hospital were obtained by
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telephone interview.
PCR assays used for detection of microbial infection of the amniotic cavity
PCR was performed for each sample using DNA extracted from both pelleted and neat amniotic fluid. Presence of M. genitalium was assessed using a previously described realtime PCR assay,7 whilst for Ureaplasma species adaptation of real-time PCR using a Taqman probe 5’-TGGAAGGTGTAGATACAATGGTTGGT-3’, allowed detection of U.
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ACCEPTED MANUSCRIPT urealyticum and/or U. parvum.8 Amplification was performed in capillaries in a total volume of 10 µl consisting of 5 µl of DNA, 1 µM each primer U4 and U5, 0.5 µM probe, 4 mM MgCl2, 1X LightCycler Faststart Master HybProbe (Roche Diagnostics, Mannheim, Germany). Using the Roche LightCycler samples were heated at 95°C for 10 min and cycled 55 times using
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parameters of 95°C for 20 sec, 55°C for 20 sec and 72°C for 30 sec. Fluorescence was
acquired once each cycle at the end of the extension segment. M. hominis was assessed
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using a previously described9, using the Roche LightCycler 480 (Roche Diagnostics,
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Mannheim, Germany).
A broad range 16S rRNA gene qPCR assay was developed in our laboratory on the LightCycler (Roche Diagnostics, Mannheim, Germany) using previously described primers fD1mod and 16S1RR-B, and 515F Taqman probe.10 The amplification reaction consisted of
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10µM of the primers fD1 mod and 16S1RR-B, 0.3 µM of Taqman probe 516F, 1X LightCycler Faststart Reaction mix (Roche), 4.5 mM MgCl2 and 1U AmpliTaq DNA Polymerase LD in a
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total reaction volume of 10 µl. An activation step of 95°C for 2 min was followed by 45 cycles at 95°C for 5 sec, 54-56°C for 10 sec increasing at increments of 0.1°C per cycle and
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60°C for 30sec, at which fluorescence was acquired once each cycle. An 18S real-time qPCR assay targeting highly conserved regions of the fungal 18S rRNA gene enabling a wide range of fungi to be detected 11 was performed as previously described.18 Stringent precautions, including the use of gamma irradiated PCR grade water, were used during the setup of broad range 16S and 18S PCR assays to avoid potential contaminants resulting in false positive results.
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ACCEPTED MANUSCRIPT RESULTS Overall, 408 women were enrolled; 375 from the Royal Women’s Hospital and 33 from the Mercy Hospital for Women. The mean age of women was 36.3 (22-47) years, with mean
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gestation at amniocentesis of 17.1 (14.6-22) weeks. Complete data on pregnancy outcomes was available for 355 women, while 53 were lost to follow-up. Eleven women underwent termination of pregnancy for an abnormal karyotype and were excluded from
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the final analysis. Of the 344 women who continued their pregnancy, 4/344 (1.2%) had an early preterm birth, prior to 34 weeks (Figure 1). One woman was delivered electively at 33
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weeks gestation for severe preeclampsia. Three women delivered spontaneously at 30, 31 and 33 weeks gestation (Table 1). These three had vaginal and placental swabs (from the amnio-chorionic membrane interface) sent for culture. The woman who delivered at 30 weeks, had a cervical cerclage inserted at 14 weeks. Whilst at delivery her vaginal swab was
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culture positive for U. urealyticum, it was not isolated from the placental, amniotic or chorionic membrane swabs. Vaginal and placental swabs were negative in the two remaining women. There were no procedure related pregnancy losses. One woman
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experienced transient amniotic fluid leak 2 days after her amniocentesis, but continued her
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pregnancy uneventfully to term.
DNA was isolated from all 688 (344 pelleted, 344 fluid) samples which were positive for the human ß-globin gene. There was a significant difference in median ß-globin level between pelleted (1,290 haploid copies/reaction) and fluid (139 haploid copies/reaction) samples (U=21.041, p=<0.001). Although all 688 samples were tested for each of the species specific assays described, no U. urealyticum, U. parvum, M. hominis or M. genitalium was detected.
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DISCUSSION
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Principal findings of the study
This study sought to determine the frequency of asymptomatic carriage of microorganisms
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in the amniotic fluid in mid trimester. We intensively investigated amniotic fluid for the presence of these microorganisms, with a particular focus on the genital mycoplasmas,
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previously reported to be associated with preterm birth.2,4,12-15 Our assessment included both sensitive species specific, and broad range bacterial and fungal PCR assays, separately examining both pelleted and neat amniotic fluid. Our principal finding is that amniotic fluid
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in mid trimester of pregnancy was found to be sterile in our population.
Moreover, our findings of no detectable micro-organisms in the amniotic cavity is consistent
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with observations of other large prospective cohorts. Studies have reported widely varying amniotic fluid positivity rates, from less than 1% to more than 80%1-3,16,17 partly explained
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by the varying techniques from culture only to single PCR targets as well as variability in clinical cohorts examined. With the addition of sophisticated molecular techniques for analysis, we have concluded that the colonization of amniotic fluid with microorganisms in the mid-trimester is rare.
Preterm labor in the absence of microbial invasion of the amniotic cavity in the mid trimester
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ACCEPTED MANUSCRIPT The observed rate of spontaneous preterm birth under 34 weeks in our study was low (3/343; 0.9%), but consistent with similar low risk populations. Notably, the pregnancies that ended in early spontaneous PTB were all negative for amniotic fluid microorganisms in the midtrimester, a finding reported by others.4 While subclinical infection has been widely
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considered to be a significant etiological factor in the pathogenesis of inflammatory preterm birth, the source of infection and gestational window at which it is acquired, remains
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unclear. It has been postulated that colonization with low virulence microorganisms, ascending from the lower genital tract, is the precursor to intra amniotic subclinical
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infection, which in turn leads to ruptured membranes and/or early preterm birth. A recent abstract published by Chu et.al. (2017) suggest an amniotic fluid microbiome exists: using a 16s rRNA MiSeq gene sequencing approach on 95 amniotic fluid samples collected for genetic testing, they report finding taxa suggestive of organisms similar to those of the
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placental membranes, the oral and nasal cavities16. Future replicate studies are required for confirmation of this however. Microorganisms commonly isolated from the genital tract, particularly the genital mycoplasmas, have been implicated in this process, yet the
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proposed mechanism by which the amniotic cavity is breached is unclear18. It is uncertain
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whether the microorganisms present are themselves pathogenic: they may be susceptibility markers or have an adjunctive role in the inflammatory preterm birth cascade. Indeed, several studies suggest that sterile inflammation is more common, and more closely associated with preterm birth than microbial associated inflammation. Gervasi et al (2012) reported an increase in markers of inflammation in the mid trimester amniotic fluid among those women destined to deliver preterm and showed that the presence of inflammation did not always reflect microbial infection, as only 0.2% of the amniotic fluid samples were culture positive.19 Recent studies using both amniotic fluid culture and broad range PCR
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ACCEPTED MANUSCRIPT with mass spectrometry have found that sterile intra-amniotic inflammation is at least as frequent, and may be more frequent, than microbial associated intra-amniotic inflammation in women with PTL.20,21 To date the presence of inflammation has been considered to be a surrogate for a microbial presence but it may be exactly the opposite; perhaps the host
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inflammatory response effects or mitigates microbial colonization and invasion. This could in part account for the range of clinical outcomes as well as differences reported in different
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populations.
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Clinical implications
While previously it has been considered that any microbial invasion of the amniotic cavity may be an important precursor to spontaneous preterm birth, the process by which this
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occurs remains elusive. This study suggests that microbial invasion of amniotic cavity is in fact uncommon. This is consistent with the findings of other large cohorts. This is noteworthy given the concept of either an amniotic fluid or placental microbiome being
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adopted16,22, as is an appreciation of its potential relevance to human pregnancy outcomes.23,24 Our study confirms that these findings should not be extrapolated to
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suggest the amniotic cavity normally contains microorganisms . Our finding of no detectable bacterial or fungal DNA in the amniotic cavity in these midtrimester asymptomatic low risk women, despite using a very comprehensive molecular approach, does not support the presence of a placental microbiome.
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ACCEPTED MANUSCRIPT That we were unable to identify any cases where amniotic fluid was positive is not surprising given the small numbers of early preterm births, but the depth of analysis undertaken in our study suggests that if amniotic cavity microbial contamination exists in these samples, it does so below the lower detection limits of the molecular assays used. All
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pregnancies that ended with early spontaneous preterm birth had no evidence of microbial contamination of the amniotic fluid in the midtrimester. While intra-amniotic infection
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remains an important contributor to spontaneous preterm birth, this study highlights that the answer does not lie solely in the search for and eradication of microorganisms, but in a
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better understanding of the activation of preterm birth inflammatory pathways.
Strengths and limitations
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Previous studies have simply used PCR to detect microbes in the amniotic fluid of midtrimester asymptomatic women, testing for one or more pre-identified microorganism.1,2,25 Strengths of this study include the prospective cohort design and the robust methodology
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employed to assess for the presence of microorganisms in the amniotic fluid. To exclude the possibility of loss of infectious agents in the supernatant following pelleting, we isolated
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DNA from both neat and pelleted amniotic fluid. The M. hominis and M. genitalium qPCR assays we utilized are very sensitive with reported lower detection limits ranging from 2 to ≥ 23 copies per reaction, respectively.9,26 The absence of specific bacteria tested for in this cohort was further supported by the negative results for the 16S assay. The lower detection limit for the 18S qPCR assay was reported as of 10 CFU/ml27 providing high sensitivity for fungal DNA, although the sensitivity of the 16S qPCR was lower (10 fold lower than the species specific assays), so bacteria in very low abundance may not have been detected.
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ACCEPTED MANUSCRIPT Nevertheless, the negative result for species specific PCR assays indicate that these microorganisms are either not present within the amniotic fluid of the women sampled during their second trimester, or that they are present at very low copy numbers, below the detection limits of these assays and are unlikely to be clinically relevant. Importantly,
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quantitative ß-globin results were positive in all samples, indicating that failure to detect microorganisms was not due to PCR inhibitors or limitations in cell adequacy, particularly for
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pelleted samples.
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While molecular microbiological laboratory data is complete for our cohort, a limitation of our study is that no culture was performed. Due to the extended recruitment phase of this study, and in particular the greater sensitivity of molecular microbiological testing compared to culture, retrospective molecular analysis only of stored frozen samples was performed.
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Moreover, clinical outcome data is not available for 53 women who delivered at their local maternity hospital, away from the recruiting centre. While this may have affected
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ascertainment of preterm birth overall, these hospitals only care for women and babies who deliver after 34 weeks gestation, so we feel that failure to detect our chosen outcome of
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early (<34 weeks) preterm birth is unlikely.
Conclusions
In our cohort study of low risk women and highly specific and sensitive broad range PCR assays of the amniotic fluid in the midtrimester has shown that it does not contain microorganisms of clinical significance at this gestation in our population.
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Acknowledgments: We would like to acknowledge Elice Rudland for her contribution to initial logging,
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processing and storage of amniotic fluid samples.
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ACCEPTED MANUSCRIPT References: 1.
Gerber S, Vial Y, Hohlfeld P, Witkin SS. Detection of Ureaplasma urealyticum in second-trimester amniotic fluid by polymerase chain reaction correlates with
2.
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subsequent preterm labor and delivery. J Infect Dis. 2003;187(3):518-521. Nguyen DP1 GS, Hohlfeld P, Sandrine G, Witkin SS. Mycoplasma hominis in midtrimester amniotic fluid: relation to pregnancy outcome. J Perinat Med.
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2004;32:323-326.
Perni SC VS, Korneeva I, Tuttle SL, Paraskevas LR, Chasen ST, Kalish RB, Witkin SS.
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Mycoplasma hominis and Ureaplasma urealyticum in midtrimester amniotic fluid: association with amniotic fluid cytokine levels and pregnancy outcome. Am J Obstet Gynecol. 2004;191(4):1382-1386. 4.
Rodriguez N, Fernandez C, Zamora Y, Berdasquera D, Rivera JA. Detection of
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Ureaplasma urealyticum and Ureaplasma parvum in amniotic fluid: association with pregnancy outcomes. The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and
Bissessor M, Tabrizi SN, Fairley CK, et al. Differing Neisseria gonorrhoeae bacterial
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Tabrizi SN, Chen S, Cohenford MA, et al. Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay. Sex Transm Infect. 2004;80(1):68-71.
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Yoshida T, Deguchi T, Ito M, Maeda S, Tamaki M, Ishiko H. Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR. J Clin Microbiol. 2002;40(4):1451-1455.
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real-time PCR for detection of Mycoplasma hominis. BMC Microbiol. 2004;4:35. Nikkari S, Lopez FA, Lepp PW, et al. Broad-range bacterial detection and the analysis of unexplained death and critical illness. Emerg Infect Dis. 2002;8(2):188-194. 11.
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Chu D SC, Seferovic M, Suter M, Cox J, Vidaeff A, Aagaard K. Profiling of microbiota in second trimester amniotic fluid reveals a distinctive community present in the mid trimester and predictive of the placental microbiome at parturition. Supplementary S1-S560 presented at 37th Annual Meeting of the Society for Maternal Fetal
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Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery.
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Gervasi MT, Romero R, Bracalente G, et al. Midtrimester amniotic fluid concentrations of interleukin-6 and interferon-gamma-inducible protein-10:
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evidence for heterogeneity of intra-amniotic inflammation and associations with spontaneous early (<32 weeks) and late (>32 weeks) preterm delivery. J Perinat Med. 2012;40(4):329-343.
Romero R, Miranda J, Chaemsaithong P, et al. Sterile and microbial-associated intra-
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amniotic inflammation in preterm prelabor rupture of membranes. J Matern Fetal Neonatal Med. 2015;28(12):1394-1409.
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Aagaard K, Ma J, Antony KM, Ganu R, Petrosino J, Versalovic J. The placenta harbors a unique microbiome. Sci Transl Med. 2014;6(237):237ra265.
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Antony KM, Ma J, Mitchell KB, Racusin DA, Versalovic J, Aagaard K. The preterm placental microbiome varies in association with excess maternal gestational weight gain. Am J Obstet Gynecol. 2015;212(5):653 e651-616.
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microbiome and pregnancy: moving beyond the vaginal microbiome. Cold Spring Harb Perspect Med. 2015;5(6).
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intra-amniotic infection in preterm labor with intact membranes. American journal of reproductive immunology. 2014;71(4):330-358. 26.
Twin J, Taylor N, Garland SM, et al. Comparison of two Mycoplasma genitalium realtime PCR detection methodologies. J Clin Microbiol. 2011;49(3):1140-1142.
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Jordanides NE, Allan EK, McLintock LA, et al. A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-
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oncology patients. Bone Marrow Transplant. 2005;35(4):389-395.
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27.
ACCEPTED MANUSCRIPT Table 1: Clinical outcomes for women presenting with early pre-term labor at <34 weeks.
Gestation at
Swab
Included in
Vaginal / Placental
study
Delivery (weeks)
CX Suture 23 weeks
Pos / Neg
Yes
( Short CX T2 Scan)
PROM 31 weeks; SPTD
33
PROM 32 weeks
33
Severe PET; CS
Neg / Neg
Yes
Neg / Neg
Yes
Neg / Neg
No*
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31
(†Pos = U. urealyticum)
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30
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Clinical Outcome
CX Suture – cervical cerclage sutures; Short CX T2 Scan – short cervix determined by transvaginal ultrasound during the second trimester; PROM - premature rupture of membranes; SPTD – spontaneous preterm delivery; PET – pre-eclampsia CS – caesarean section; †HVS – high vaginal swab positive for Ureaplasma urealyticum. *excluded from analysis due to severe pre-eclampsia resulting in delivery prior to 34 weeks.
ACCEPTED MANUSCRIPT Figure 1. Flowchart outlining study recruitment, design and pregnancy outcomes.
Royal Women’’s
Mercy Hospital for Women
Hospital
Amniotic fluid samples obtained
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Procedural related loss
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n=408
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n=33
n=375
Pregnancies ongoing
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n=408
Pregnancies ongoing
Termination of pregnancy for genetic abnormality n=11
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n=397
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Women lost to follow-up n=53
Women for whom pregnancy outcome data is available n=344
Pre-term delivery <34
Preterm delivery rate
weeks gestation
1.2%
n=4
S0002-9378(17)30383-6
DOI:
10.1016/j.ajog.2017.02.051
Reference:
YMOB 11571
To appear in:
American Journal of Obstetrics and Gynecology
Received Date: 21 February 2017 Revised Date:
26 February 2017
Accepted Date: 27 February 2017
Please cite this article as: Rowlands S, Danielewski JA, Tabrizi SN, Walker SP, Garland SM, Microbial Invasion of the Amniotic Cavity in Mid-Trimester Pregnancies Using Molecular Microbiology, American Journal of Obstetrics and Gynecology (2017), doi: 10.1016/j.ajog.2017.02.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT Microbial invasion of the amniotic cavity in mid-trimester pregnancies using molecular microbiology.
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Shelley ROWLANDS1,2, MD; Jennifer A. DANIELEWSKI3, PhD; Sepehr N. TABRIZI2,3, PhD; Susan
1
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P. WALKER4,5, MD; Suzanne M. GARLAND2,3, MD
Department of Maternity Services, Epworth Freemasons Hospital, East Melbourne, 3002
2
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Victoria, Australia.
Department of Obstetrics and Gynaecology, University of Melbourne,
Royal Women’s Hospital, Parkville, 3052 Victoria, Australia.
Department of Microbiology and Infectious Diseases, Murdoch Childrens Research
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Institute, Parkville, 3052, Victoria, Australia.
Department of Perinatal Medicine, Mercy Hospital for Women, Heidelberg, 3084, Victoria,
Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for
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Women, Heidelberg, 3084, Victoria, Australia
Word Count: Abstract 209, Main text 2741
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ACCEPTED MANUSCRIPT Funding source: This study was supported in part by the Royal Women's Hospital Education fund and the
Conflict of Interest / Disclosure statement
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The authors report no conflict of interest.
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Microbiology Infectious Diseases Research fund.
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Corresponding Author: Professor Suzanne Garland, Department of Microbiology and Infectious Diseases, The Royal Women's Hospital, Locked Bag 300, Parkville, 3052, Victoria, Australia.
Fax: + 61-3-9347 8235
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Phone: + 61-3-8345 3670
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Condensation Subclinical colonization of the amniotic cavity with microorganisms is rare in the mid trimester among low risk women. In 344 women between 15 and 22 weeks of gestation no
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microbial invasion of the amniotic cavity was detected using species specific PCRs, as well as 16s and 18s approaches.
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Short version of title
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Microorganisms were not detected in the amniotic fluid in mid-trimester of pregnancy
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ACCEPTED MANUSCRIPT ABSTRACT Objective To determine the frequency of microbial invasion of the amniotic cavity in the midtrimester
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of pregnancy in patients undergoing amniocentesis for clinical indications. Study Design
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Prospective investigation of the amniotic fluid of 344 asymptomatic women recruited in mid pregnancy for the presence of microbial DNA. Amniotic samples obtained at time of
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amniocentesis for genetic testing on women between 15-22 weeks gestation were tested specifically for the presence of Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium, as well as for other bacteria and fungi using broadrange PCR only. Pregnancy outcomes were reviewed independent of all molecular test
Results
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results.
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Using broad-range PCR, the prevalence of microbial invasion of the amniotic cavity in women between 15 and 22 weeks of gestation was 0% (0 vs. 344). Early preterm delivery
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occurred in only 4 (1%) women; 1 delivered electively and 3 spontaneously. None were associated with U. urealyticum, U.parvum, M.hominis, or M. genitalium. In addition, broad range PCR did not reveal the presence of other bacterial or fungal microbes. Conclusions Microbial invasion of the amniotic cavity in midtrimester gestations of low-risk pregnant women was not detected using molecular methods in 344 patients.
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ACCEPTED MANUSCRIPT Keywords Preterm birth, mid-trimester, amniotic fluid, intraamniotic infection, chorioamnionitis,
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sterile biological fluid, molecular microbiology, fungi, mycoplasma, ureaplasma.
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ACCEPTED MANUSCRIPT INTRODUCTION Studies have investigated the presence of microorganisms in amniotic fluid among asymptomatic women from amniocentesis performed in the mid-trimester for genetic screening purposes. Some,1-3 but not all4 studies have suggested that U. urealyticum or M.
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hominis in the amniotic fluid may be associated with an increased risk for preterm pre-labor rupture of membranes (PPROM), preterm labor (PTL) and/or delivery (PTD). The role of
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Mycoplasma species and their clinical significance in the amniotic fluid of asymptomatic women remains unresolved. Further studies are needed to understand the aetiology and
amniotic microbial invasion.
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pathogenesis of preterm labor in relation to the presence or absence of asymptomatic intra-
We conducted a longitudinal study on a low risk cohort of healthy asymptomatic women, to
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determine the prevalence of intra- amniotic microbial invasion and to examine their subsequent pregnancy outcomes. Amniotic fluid sampled between 15 and 22 weeks
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gestation was assessed for the presence of microorganisms in the amniotic cavity, by species specific PCR for U. urealyticum, U. parvum, M. hominis and M. genitalium, and
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presence of other bacterial and fungal pathogens using broad-range 16S and 18S PCR, respectively.
MATERIALS AND METHODS Patient population Women were recruited from two major tertiary referral centres in Melbourne, Australia: the
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ACCEPTED MANUSCRIPT Royal Women’s Hospital, and The Mercy Hospital for Women, between September 2004 and December 2008. Study inclusion criteria were women undergoing amniocentesis for genetic prenatal diagnosis between 15-22 weeks gestation in a singleton pregnancy. Women were excluded if a fetal abnormality were identified on ultrasound or if they had
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current clinical evidence of infection (of any type) for which they had received antibiotic treatment within the preceding two weeks. Consecutive women were approached and the
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majority agreed to participate in the study. Gestation was established by ultrasound scan between 8-14 weeks. All women provided written informed consent. Ethics approval was
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received from the Research and Ethics Committees at both hospitals.
Sample processing
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Amniotic fluid collected at the time of amniocentesis was stored at -80oC until all samples were collected and then processed over the following one to two years. Processing involved two different preparation methods prior to DNA isolation: (i) concentration of amniotic
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fluid whereby one ml was centrifuged at 14,000x g for 15 minutes to ensure all biological particles were captured, with the resultant pellet resuspended in 200 µl of phosphate
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buffered saline; and (ii) 200 µl of neat (undiluted, non-centrifuged) amniotic fluid. The automated MagNA Pure LC extraction system (Roche Diagnostics, Mannheim, Germany) was used to isolate DNA with the MagNA Pure LC DNA Isolation Kit I on amniotic fluid preparations (i) and (ii). To improve the extraction efficiency, 33.3µg/ml of poly(A) RNA carrier was added to the lysis buffer, due to the potential for low concentrations of bacterial and fungal DNA in these samples. Nucleic acid was eluted into a final volume of 100 µl. To
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ACCEPTED MANUSCRIPT determine cell adequacy, and as an internal control for extraction and amplification procedures, qPCR targeting a 260 bp region of the human ß-globin gene was performed.5,6
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Clinical definitions and outcomes Microbial invasion of the amniotic cavity was defined as the detection of any bacterial or fungal microorganism in the amniotic fluid using the species specific and broad range 16S
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and 18S assays outlined below. In assessing subsequent pregnancy outcomes; early
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preterm birth was defined as birth occurring prior to 34 completed weeks’ gestation, preterm labour was defined as regular uterine contractions and cervical change at <34 completed weeks gestation, where intervention such as tocolysis, cervical cerclage or administration of prophylactic steroids for fetal lung maturation were considered to be
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indicated and PPROM was defined as spontaneous rupture of membranes prior to the onset of labor and before 34 completed weeks’ gestation. Medical record reviews were used to determine delivery outcomes for women who delivered at the recruiting hospitals, while
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outcomes for women who delivered at a non-recruiting hospital were obtained by
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telephone interview.
PCR assays used for detection of microbial infection of the amniotic cavity
PCR was performed for each sample using DNA extracted from both pelleted and neat amniotic fluid. Presence of M. genitalium was assessed using a previously described realtime PCR assay,7 whilst for Ureaplasma species adaptation of real-time PCR using a Taqman probe 5’-TGGAAGGTGTAGATACAATGGTTGGT-3’, allowed detection of U.
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ACCEPTED MANUSCRIPT urealyticum and/or U. parvum.8 Amplification was performed in capillaries in a total volume of 10 µl consisting of 5 µl of DNA, 1 µM each primer U4 and U5, 0.5 µM probe, 4 mM MgCl2, 1X LightCycler Faststart Master HybProbe (Roche Diagnostics, Mannheim, Germany). Using the Roche LightCycler samples were heated at 95°C for 10 min and cycled 55 times using
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parameters of 95°C for 20 sec, 55°C for 20 sec and 72°C for 30 sec. Fluorescence was
acquired once each cycle at the end of the extension segment. M. hominis was assessed
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using a previously described9, using the Roche LightCycler 480 (Roche Diagnostics,
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Mannheim, Germany).
A broad range 16S rRNA gene qPCR assay was developed in our laboratory on the LightCycler (Roche Diagnostics, Mannheim, Germany) using previously described primers fD1mod and 16S1RR-B, and 515F Taqman probe.10 The amplification reaction consisted of
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10µM of the primers fD1 mod and 16S1RR-B, 0.3 µM of Taqman probe 516F, 1X LightCycler Faststart Reaction mix (Roche), 4.5 mM MgCl2 and 1U AmpliTaq DNA Polymerase LD in a
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total reaction volume of 10 µl. An activation step of 95°C for 2 min was followed by 45 cycles at 95°C for 5 sec, 54-56°C for 10 sec increasing at increments of 0.1°C per cycle and
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60°C for 30sec, at which fluorescence was acquired once each cycle. An 18S real-time qPCR assay targeting highly conserved regions of the fungal 18S rRNA gene enabling a wide range of fungi to be detected 11 was performed as previously described.18 Stringent precautions, including the use of gamma irradiated PCR grade water, were used during the setup of broad range 16S and 18S PCR assays to avoid potential contaminants resulting in false positive results.
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ACCEPTED MANUSCRIPT RESULTS Overall, 408 women were enrolled; 375 from the Royal Women’s Hospital and 33 from the Mercy Hospital for Women. The mean age of women was 36.3 (22-47) years, with mean
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gestation at amniocentesis of 17.1 (14.6-22) weeks. Complete data on pregnancy outcomes was available for 355 women, while 53 were lost to follow-up. Eleven women underwent termination of pregnancy for an abnormal karyotype and were excluded from
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the final analysis. Of the 344 women who continued their pregnancy, 4/344 (1.2%) had an early preterm birth, prior to 34 weeks (Figure 1). One woman was delivered electively at 33
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weeks gestation for severe preeclampsia. Three women delivered spontaneously at 30, 31 and 33 weeks gestation (Table 1). These three had vaginal and placental swabs (from the amnio-chorionic membrane interface) sent for culture. The woman who delivered at 30 weeks, had a cervical cerclage inserted at 14 weeks. Whilst at delivery her vaginal swab was
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culture positive for U. urealyticum, it was not isolated from the placental, amniotic or chorionic membrane swabs. Vaginal and placental swabs were negative in the two remaining women. There were no procedure related pregnancy losses. One woman
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experienced transient amniotic fluid leak 2 days after her amniocentesis, but continued her
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pregnancy uneventfully to term.
DNA was isolated from all 688 (344 pelleted, 344 fluid) samples which were positive for the human ß-globin gene. There was a significant difference in median ß-globin level between pelleted (1,290 haploid copies/reaction) and fluid (139 haploid copies/reaction) samples (U=21.041, p=<0.001). Although all 688 samples were tested for each of the species specific assays described, no U. urealyticum, U. parvum, M. hominis or M. genitalium was detected.
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DISCUSSION
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Principal findings of the study
This study sought to determine the frequency of asymptomatic carriage of microorganisms
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in the amniotic fluid in mid trimester. We intensively investigated amniotic fluid for the presence of these microorganisms, with a particular focus on the genital mycoplasmas,
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previously reported to be associated with preterm birth.2,4,12-15 Our assessment included both sensitive species specific, and broad range bacterial and fungal PCR assays, separately examining both pelleted and neat amniotic fluid. Our principal finding is that amniotic fluid
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in mid trimester of pregnancy was found to be sterile in our population.
Moreover, our findings of no detectable micro-organisms in the amniotic cavity is consistent
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with observations of other large prospective cohorts. Studies have reported widely varying amniotic fluid positivity rates, from less than 1% to more than 80%1-3,16,17 partly explained
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by the varying techniques from culture only to single PCR targets as well as variability in clinical cohorts examined. With the addition of sophisticated molecular techniques for analysis, we have concluded that the colonization of amniotic fluid with microorganisms in the mid-trimester is rare.
Preterm labor in the absence of microbial invasion of the amniotic cavity in the mid trimester
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ACCEPTED MANUSCRIPT The observed rate of spontaneous preterm birth under 34 weeks in our study was low (3/343; 0.9%), but consistent with similar low risk populations. Notably, the pregnancies that ended in early spontaneous PTB were all negative for amniotic fluid microorganisms in the midtrimester, a finding reported by others.4 While subclinical infection has been widely
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considered to be a significant etiological factor in the pathogenesis of inflammatory preterm birth, the source of infection and gestational window at which it is acquired, remains
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unclear. It has been postulated that colonization with low virulence microorganisms, ascending from the lower genital tract, is the precursor to intra amniotic subclinical
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infection, which in turn leads to ruptured membranes and/or early preterm birth. A recent abstract published by Chu et.al. (2017) suggest an amniotic fluid microbiome exists: using a 16s rRNA MiSeq gene sequencing approach on 95 amniotic fluid samples collected for genetic testing, they report finding taxa suggestive of organisms similar to those of the
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placental membranes, the oral and nasal cavities16. Future replicate studies are required for confirmation of this however. Microorganisms commonly isolated from the genital tract, particularly the genital mycoplasmas, have been implicated in this process, yet the
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proposed mechanism by which the amniotic cavity is breached is unclear18. It is uncertain
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whether the microorganisms present are themselves pathogenic: they may be susceptibility markers or have an adjunctive role in the inflammatory preterm birth cascade. Indeed, several studies suggest that sterile inflammation is more common, and more closely associated with preterm birth than microbial associated inflammation. Gervasi et al (2012) reported an increase in markers of inflammation in the mid trimester amniotic fluid among those women destined to deliver preterm and showed that the presence of inflammation did not always reflect microbial infection, as only 0.2% of the amniotic fluid samples were culture positive.19 Recent studies using both amniotic fluid culture and broad range PCR
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ACCEPTED MANUSCRIPT with mass spectrometry have found that sterile intra-amniotic inflammation is at least as frequent, and may be more frequent, than microbial associated intra-amniotic inflammation in women with PTL.20,21 To date the presence of inflammation has been considered to be a surrogate for a microbial presence but it may be exactly the opposite; perhaps the host
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inflammatory response effects or mitigates microbial colonization and invasion. This could in part account for the range of clinical outcomes as well as differences reported in different
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populations.
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Clinical implications
While previously it has been considered that any microbial invasion of the amniotic cavity may be an important precursor to spontaneous preterm birth, the process by which this
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occurs remains elusive. This study suggests that microbial invasion of amniotic cavity is in fact uncommon. This is consistent with the findings of other large cohorts. This is noteworthy given the concept of either an amniotic fluid or placental microbiome being
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adopted16,22, as is an appreciation of its potential relevance to human pregnancy outcomes.23,24 Our study confirms that these findings should not be extrapolated to
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suggest the amniotic cavity normally contains microorganisms . Our finding of no detectable bacterial or fungal DNA in the amniotic cavity in these midtrimester asymptomatic low risk women, despite using a very comprehensive molecular approach, does not support the presence of a placental microbiome.
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ACCEPTED MANUSCRIPT That we were unable to identify any cases where amniotic fluid was positive is not surprising given the small numbers of early preterm births, but the depth of analysis undertaken in our study suggests that if amniotic cavity microbial contamination exists in these samples, it does so below the lower detection limits of the molecular assays used. All
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pregnancies that ended with early spontaneous preterm birth had no evidence of microbial contamination of the amniotic fluid in the midtrimester. While intra-amniotic infection
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remains an important contributor to spontaneous preterm birth, this study highlights that the answer does not lie solely in the search for and eradication of microorganisms, but in a
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better understanding of the activation of preterm birth inflammatory pathways.
Strengths and limitations
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Previous studies have simply used PCR to detect microbes in the amniotic fluid of midtrimester asymptomatic women, testing for one or more pre-identified microorganism.1,2,25 Strengths of this study include the prospective cohort design and the robust methodology
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employed to assess for the presence of microorganisms in the amniotic fluid. To exclude the possibility of loss of infectious agents in the supernatant following pelleting, we isolated
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DNA from both neat and pelleted amniotic fluid. The M. hominis and M. genitalium qPCR assays we utilized are very sensitive with reported lower detection limits ranging from 2 to ≥ 23 copies per reaction, respectively.9,26 The absence of specific bacteria tested for in this cohort was further supported by the negative results for the 16S assay. The lower detection limit for the 18S qPCR assay was reported as of 10 CFU/ml27 providing high sensitivity for fungal DNA, although the sensitivity of the 16S qPCR was lower (10 fold lower than the species specific assays), so bacteria in very low abundance may not have been detected.
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ACCEPTED MANUSCRIPT Nevertheless, the negative result for species specific PCR assays indicate that these microorganisms are either not present within the amniotic fluid of the women sampled during their second trimester, or that they are present at very low copy numbers, below the detection limits of these assays and are unlikely to be clinically relevant. Importantly,
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quantitative ß-globin results were positive in all samples, indicating that failure to detect microorganisms was not due to PCR inhibitors or limitations in cell adequacy, particularly for
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pelleted samples.
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While molecular microbiological laboratory data is complete for our cohort, a limitation of our study is that no culture was performed. Due to the extended recruitment phase of this study, and in particular the greater sensitivity of molecular microbiological testing compared to culture, retrospective molecular analysis only of stored frozen samples was performed.
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Moreover, clinical outcome data is not available for 53 women who delivered at their local maternity hospital, away from the recruiting centre. While this may have affected
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ascertainment of preterm birth overall, these hospitals only care for women and babies who deliver after 34 weeks gestation, so we feel that failure to detect our chosen outcome of
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early (<34 weeks) preterm birth is unlikely.
Conclusions
In our cohort study of low risk women and highly specific and sensitive broad range PCR assays of the amniotic fluid in the midtrimester has shown that it does not contain microorganisms of clinical significance at this gestation in our population.
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Acknowledgments: We would like to acknowledge Elice Rudland for her contribution to initial logging,
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processing and storage of amniotic fluid samples.
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ACCEPTED MANUSCRIPT References: 1.
Gerber S, Vial Y, Hohlfeld P, Witkin SS. Detection of Ureaplasma urealyticum in second-trimester amniotic fluid by polymerase chain reaction correlates with
2.
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subsequent preterm labor and delivery. J Infect Dis. 2003;187(3):518-521. Nguyen DP1 GS, Hohlfeld P, Sandrine G, Witkin SS. Mycoplasma hominis in midtrimester amniotic fluid: relation to pregnancy outcome. J Perinat Med.
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Mycoplasma hominis and Ureaplasma urealyticum in midtrimester amniotic fluid: association with amniotic fluid cytokine levels and pregnancy outcome. Am J Obstet Gynecol. 2004;191(4):1382-1386. 4.
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Ureaplasma urealyticum and Ureaplasma parvum in amniotic fluid: association with pregnancy outcomes. The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and
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Yoshida T, Deguchi T, Ito M, Maeda S, Tamaki M, Ishiko H. Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR. J Clin Microbiol. 2002;40(4):1451-1455.
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real-time PCR for detection of Mycoplasma hominis. BMC Microbiol. 2004;4:35. Nikkari S, Lopez FA, Lepp PW, et al. Broad-range bacterial detection and the analysis of unexplained death and critical illness. Emerg Infect Dis. 2002;8(2):188-194. 11.
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Chu D SC, Seferovic M, Suter M, Cox J, Vidaeff A, Aagaard K. Profiling of microbiota in second trimester amniotic fluid reveals a distinctive community present in the mid trimester and predictive of the placental microbiome at parturition. Supplementary S1-S560 presented at 37th Annual Meeting of the Society for Maternal Fetal
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Gervasi MT, Romero R, Bracalente G, et al. Midtrimester amniotic fluid concentrations of interleukin-6 and interferon-gamma-inducible protein-10:
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evidence for heterogeneity of intra-amniotic inflammation and associations with spontaneous early (<32 weeks) and late (>32 weeks) preterm delivery. J Perinat Med. 2012;40(4):329-343.
Romero R, Miranda J, Chaemsaithong P, et al. Sterile and microbial-associated intra-
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amniotic inflammation in preterm prelabor rupture of membranes. J Matern Fetal Neonatal Med. 2015;28(12):1394-1409.
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Antony KM, Ma J, Mitchell KB, Racusin DA, Versalovic J, Aagaard K. The preterm placental microbiome varies in association with excess maternal gestational weight gain. Am J Obstet Gynecol. 2015;212(5):653 e651-616.
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intra-amniotic infection in preterm labor with intact membranes. American journal of reproductive immunology. 2014;71(4):330-358. 26.
Twin J, Taylor N, Garland SM, et al. Comparison of two Mycoplasma genitalium realtime PCR detection methodologies. J Clin Microbiol. 2011;49(3):1140-1142.
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Jordanides NE, Allan EK, McLintock LA, et al. A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-
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oncology patients. Bone Marrow Transplant. 2005;35(4):389-395.
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ACCEPTED MANUSCRIPT Table 1: Clinical outcomes for women presenting with early pre-term labor at <34 weeks.
Gestation at
Swab
Included in
Vaginal / Placental
study
Delivery (weeks)
CX Suture 23 weeks
Pos / Neg
Yes
( Short CX T2 Scan)
PROM 31 weeks; SPTD
33
PROM 32 weeks
33
Severe PET; CS
Neg / Neg
Yes
Neg / Neg
Yes
Neg / Neg
No*
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31
(†Pos = U. urealyticum)
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30
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Clinical Outcome
CX Suture – cervical cerclage sutures; Short CX T2 Scan – short cervix determined by transvaginal ultrasound during the second trimester; PROM - premature rupture of membranes; SPTD – spontaneous preterm delivery; PET – pre-eclampsia CS – caesarean section; †HVS – high vaginal swab positive for Ureaplasma urealyticum. *excluded from analysis due to severe pre-eclampsia resulting in delivery prior to 34 weeks.
ACCEPTED MANUSCRIPT Figure 1. Flowchart outlining study recruitment, design and pregnancy outcomes.
Royal Women’’s
Mercy Hospital for Women
Hospital
Amniotic fluid samples obtained
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Procedural related loss
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n=408
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n=33
n=375
Pregnancies ongoing
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n=408
Pregnancies ongoing
Termination of pregnancy for genetic abnormality n=11
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n=397
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Women lost to follow-up n=53
Women for whom pregnancy outcome data is available n=344
Pre-term delivery <34
Preterm delivery rate
weeks gestation
1.2%
n=4