Bogaert, Debby

de Steenhuijsen Piters, Wouter A A

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2. Mills NL, Miller JJ, Anand A, Robinson SD, Frazer GA, Anderson D, et al.Increased arterial stiffness in patients with chronic obstructivepulmonary disease: a mechanism for increased cardiovascular risk.Thorax 2008;63:306–311.3. Dransfield MT, Cockcroft JR, Townsend RR, Coxson HO, Sharma SS,Rubin DB, et al. Effect of fluticasone propionate/salmeterol onarterial stiffness in patients with COPD. Respir Med 2011;105:1322–1330.4. Pepin JL, Cockcroft JR, Midwinter D, Sharma S, Rubin DB, Andreas S.Long-acting bronchodilators and arterial stiffness in patients withCOPD: a comparison of fluticasone furoate/vilanterol with tiotropium.Chest 2014;146:1521–1530.5. Bhatt SP, Dransfield MT, Cockcroft JR, Wang-Jairaj J, Midwinter DA,Rubin DB, et al. A randomized trial of once-daily fluticasonefuroate/vilanterol or vilanterol versus placebo to determine effects onarterial stiffness in COPD. Int J Chron Obstruct Pulmon Dis 2017;12:351–365.6. Vestbo J, Anderson JA, Brook RD, Calverley PMA, Celli BR, Crim C,et al.; SUMMIT Investigators. Fluticasone furoate and vilanterol andsurvival in chronic obstructive pulmonary disease with heightenedcardiovascular risk (SUMMIT): a double-blind randomised controlledtrial. Lancet 2016;387:1817–1826.7. Boutouyrie P, Vermeersch SJ; Reference Values for Arterial Stiffness’Collaboration. Determinants of pulse wave velocity in healthy peopleand in the presence of cardiovascular risk factors: ‘establishingnormal and reference values’. Eur Heart J 2010;31:2338–2350.8. Sin DD, Man SFP. Chronic obstructive pulmonary disease as a riskfactor for cardiovascular morbidity and mortality. Proc Am Thorac Soc2005;2:8–11.9. Agustı́ A, Edwards LD, Rennard SI, MacNee W, Tal-Singer R, Miller BE,et al.; Evaluation of COPD Longitudinally to Identify PredictiveSurrogate Endpoints (ECLIPSE) Investigators. Persistent systemicinflammation is associated with poor clinical outcomes in COPD: anovel phenotype. PLoS One 2012;7:e37483.10. Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, Guize L, et al.Aortic stiffness is an independent predictor of all-cause andcardiovascular mortality in hypertensive patients. Hypertension2001;37:1236–1241.11. McAllister DA, Maclay JD, Mills NL, Mair G, Miller J, Anderson D,et al. Arterial stiffness is independently associated withemphysema severity in patients with chronic obstructivepulmonary disease. Am J Respir Crit Care Med 2007;176:1208–1214.Copyright © 2020 by the American Thoracic SocietyBacterial DNA in Fetal Lung SamplesMay Be Explainedby Sample ContaminationTo the Editor:We read with interest the work of Al Alam and colleagues (1)suggesting the existence of a fetal lung microbiome signature. Asstated by the authors, the existence of a fetal microbiome remains amatter of debate. Collecting fetal material from human subjectsduring pregnancy is difficult, and therefore studies so far havemainly sampled placenta and amniotic fluid as proxies for thefetomaternal interface. Detection of microbial communities inthese samples would support the existence of a fetal microbiome,which would open up the possibility of microbe-drivenimmune training commencing before birth.In a recently published study, de Goffau and colleaguesrefuted the existence of a placental microbiome in 578 placentalsamples from both complicated and uncomplicated pregnancies,with the exception of Streptococcus agalactiae, which was foundin z5% of the samples (2). Using a rigorous approachthat included analyses of extensive negative and positivecontrols, DNA-sequencing techniques, and the use of differentDNA-isolation kits, they disproved previous claims of theexistence of a placental microbiome (3) and suggested thatcontamination issues provide a convincing explanation forearlier findings.Because the current study by Al Alam and colleagues (1),which describes microbial presence in 31 fetal/placental samplescollected between 11 and 20 weeks of gestation after dilatationand curettage (D&C) and dilatation and evacuation (D&E)procedures, contradicts the results obtained by de Goffau andcolleagues (2), we attempted to identify potential explanationsfor these discrepancies.First, given the low biomass observed in fetal (lung)samples (1), the risk of contamination from nonbiological sourcesis high. Contamination may occur during sample collection, asboth D&C and D&E procedures require passage of a medicalinstrument through the vagina, which is known to harbor adense microbiota. Subsequent technical procedures can furthercontaminate samples, for example, by DNA-isolation reagents,well-to-well contamination, and the sequencing machine itself (2).Some contamination risks are unavoidable; however, toappropriately control for these risks, we would have expected morerigorous technical controls at various stages of the laboratoryprocess, instead of the limited number of controls (n= 2) obtainedfrom only one part of the lab procedures of one of the twolaboratories involved. It would have been even more important tocontrol for contamination during sample collection, by collectingcontrol samples from the medical equipment and the localenvironment before and during the D&C or D&E procedure;however, the authors do not refer to such controls.The reported fetal lung profiles obtained by 16S ribosomal RNAamplicon sequencing are very biodiverse and include speciespreviously described as part of the so-called “kitome” (i.e.,contaminating DNA present in DNA-extraction reagents) (4),suggesting that the reported results can at least partly be explainedby contamination. This notion is further supported by the reporteddiscrepancy between 16S-based and whole-genome shotgunsequencing, with the latter showing no bacterial signature at all.In addition, the comparison between microbial DNA observedin samples versus controls (Figure 1B of Reference 1) suggests thepossibility of vaginal cross-contamination, as highly abundantgenera in the vaginal tract, Lactobacillus and Gardnerella, arealso the most abundant genera reported in fetal samples.This article is open access and distributed under the terms of the CreativeCommons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). For commercial usage andreprints, please contact Diane Gern (dgern@thoracic.org).Originally Published in Press as DOI: 10.1164/rccm.202001-0050LE onFebruary 12, 2020CORRESPONDENCE1310 American Journal of Respiratory and Critical Care Medicine Volume 201 Number 10 | May 15 2020 In addition, the lack of information about the indicationsfor the D&C or D&E procedures makes us question whetherpregnancy complications, such as miscarriage, which islikely accompanied by local inflammation (5), bacterialtranslocation, and infection, might be at the basis of theirfindings. All of the above information is essential to assessthe potential biological origin of the observed bacterial DNAin fetal samples.In conclusion, although we do not dispute the possibility of theexistence of a fetal lung microbiome signature, the study lackedrobust controls during both sample collection and laboratoryprocesses, giving rise to speculation about the validity of thereported findings. Therefore, we believe that the presented datainsufficiently support the authors’ conclusion that the human fetallung harbors a microbiome signature. We do agree, however, thatthe road on which they have embarked—studying fetal tissues atearly stages of pregnancy—is an interesting and important one, andtherefore warrants more research. nAuthor disclosures are available with the text of this letter atwww.atsjournals.org.Wouter A. A. de Steenhuijsen Piters, M.D., Ph.D.*Wilhelmina Children’s Hospital/University Medical Center UtrechtUtrecht, the NetherlandsandNational Institute for Public Health and the EnvironmentBilthoven, the NetherlandsDebby Bogaert, M.D., Ph.D.Wilhelmina Children’s Hospital/University Medical Center UtrechtUtrecht, the NetherlandsNational Institute for Public Health and the EnvironmentBilthoven, the NetherlandsandUniversity of EdinburghEdinburgh, United KingdomORCID IDs: 0000-0002-1144-4067 (W.A.A.d.S.P.);0000-0003-2651-7000 (D.B.).*Corresponding author (e-mail: w.a.a.desteenhuijsenpiters@umcutrecht.nl).References1. Al Alam D, Danopoulos S, Grubbs B, Ali NABM, MacAogain M,Chotirmall SH, et al. Human fetal lungs harbor a microbiome signature[letter]. Am J Respir Crit Care Med 2020;201:1002–1006.2. de Goffau MC, Lager S, Sovio U, Gaccioli F, Cook E,Peacock SJ, et al. Human placenta has no microbiomebut can contain potential pathogens. Nature 2019;572:329–334.3. Aagaard K, Ma J, Antony KM, Ganu R, Petrosino J, Versalovic J. Theplacenta harbors a unique microbiome. Sci Transl Med 2014;6:237ra65.4. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, et al.Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 2014;12:87.5. Arck PC, Hecher K. Fetomaternal immune cross-talk and itsconsequences for maternal and offspring’s health. Nat Med 2013;19:548–556.Copyright © 2020 by the American Thoracic SocietyReply to de Steenhuijsen Piters and BogaertFrom the Authors:We appreciate the thoughtful letter from de Steenhuijsen Pitersand Bogaert in response to our recent report (1). Our proof-of-concept study is the first demonstration that a microbiomesignature can be identified in human fetal lung tissues. Newernext-generation culture-independent methodologies, howeverpromising, present numerous challenges and questions, especiallywith regard to issues involving methodology. Hence, weappreciate the opportunity to address and expand upon ourfindings, and we welcome a scientific discussion to provide someclarifications or answers.We concur with de Steenhuijsen Piters and Bogaert thatcollecting fetal material from human subjects is extremelychallenging, and pregnancy complications such as miscarriagecould be accompanied by local inflammation. Nevertheless, inour study, all samples were collected from assumed normal subjectsafter an elective abortion and after informed consent was obtained.No samples were derived from a miscarriage, and thereforethe possibility of inflammation/bacterial translocation due tomiscarriage or membrane rupture is extremely low.Given the complication of host DNA contaminationcoupled with low microbial biomass, these samples were indeedchallenging to work with, as alluded to by de Steenhuijsen Piters andBogaert and others (2). At the time of sample collection, just as withany routine medical procedure, all instruments were sterilizedbefore each procedure (dilatation and curettage or dilatation andevacuation) in each patient. This was the first study to sample bothplacenta and deep fetal tissue (intestine and lung), and we agreewith the concerns about the likelihood of possible contaminationfrom nonbiological sources in low-biomass samples such as these.The abortion material was extracted through the vaginal canal,which is not sterile, and some of the material could have come incontact with the vaginal flora. However, our data showedstatistically distinct signatures in the lungs and placenta from thesame subjects, suggesting that the DNA signature was specific andnot just the result of detecting a contaminant from the vaginalcanal. Samples from twin pairs in the cohort also had differentmicrobiome signatures even though they passed through the samevaginal canal. Moreover, a recent study showed no differences inthe microbiome signature in placenta, fetal membranes, ormeconium between vaginal and Caesarian-section deliveries (3). Inaddition, a study from our group demonstrated the presence ofabundant immune cell signatures in the fetal lung as early as 11This article is open access and distributed under the terms of the CreativeCommons Attribution Non-Commercial No Derivatives License 4.0(http://creativecommons.org/licenses/by-nc-nd/4.0/). For commercialusage and reprints, please contact Diane Gern (dgern@thoracic.org).Supported by NIH grant K08HL141652 (C.V.L.).Author Contributions: D.A.A., S.D., N.A’t.B.M.A., M.M.A., S.H.C., D.W.,A.G., N.A., and C.V.L. drafted, edited, and approved the letter. B.G. drafted,edited, and approved the letter.Originally Published in Press as DOI: 10.1164/rccm.202001-0221LE onFebruary 12, 2020CORRESPONDENCECorrespondence 1311 

Bacterial DNA in Fetal Lung Samples May Be Explained by Sample Contamination

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Bacterial DNA in Fetal Lung Samples May Be Explained by Sample Contamination

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