Midtrimester amniotic fluid from healthy pregnancies has no microorganisms using multiple methods of microbiologic inquiry

Journal Pre-proof Midtrimester amniotic fluid from healthy pregnancies has no microorganisms using multiple methods of microbiologic inquiry Yu Liu, PhD, Xiang Li, PhD, Baoli Zhu, PhD, Haijuan Zhao, MS, Qubo Ai, MS, Yulong Tong, MS, Shengtang Qin, PhD, Ye Feng, MD, Yutong Wang, MD, Shuxian Wang, PhD, Jingmei Ma, MD, PhD, Huixia Yang, MD, PhD PII:

S0002-9378(20)30111-3

DOI:

https://doi.org/10.1016/j.ajog.2020.01.056

Reference:

YMOB 13097

To appear in:

American Journal of Obstetrics and Gynecology

Received Date: 29 October 2019 Revised Date:

27 January 2020

Accepted Date: 27 January 2020

Please cite this article as: Liu Y, Li X, Zhu B, Zhao H, Ai Q, Tong Y, Qin S, Feng Y, Wang Y, Wang S, Ma J, Yang H, Midtrimester amniotic fluid from healthy pregnancies has no microorganisms using multiple methods of microbiologic inquiry, American Journal of Obstetrics and Gynecology (2020), doi: https://doi.org/10.1016/j.ajog.2020.01.056. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc.

1

Midtrimester amniotic fluid from healthy pregnancies has no

2

microorganisms using multiple methods of microbiologic inquiry

3 4

Yu Liu, PhD1,2, Xiang Li, PhD3, Baoli Zhu, PhD4, Haijuan Zhao, MS3, Qubo Ai, MS3,

5

Yulong Tong, MS1,2, Shengtang Qin, PhD1,2, Ye Feng, MD1,2, Yutong Wang, MD1,2,

6

Shuxian Wang, PhD1,2, Jingmei Ma, MD, PhD1,2, Huixia Yang, MD, PhD1,2

7 8

1

9

China.

Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing,

10

2

11

Beijing, China.

12

3

13

4

14

Microbiology, Chinese Academy of Science, Beijing, China.

Beijing Key Laboratory of Maternal Fetal Medicine of Gestational Diabetes Mellitus,

COYOTE Medical Laboratory, Beijing, China. Key Laboratory of Pathogenic Microbiology and Immunology/ Institute of

15 16

Declaration of interests: The authors report no conflicts of interest.

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Corresponding authors: Jingmei Ma, [email protected]

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Huixia Yang, [email protected]

20 21

Funding: The research was financially supported by the National Key

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Technologies R&D Program (no. 2016YFC1000303), National Natural

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Science Foundation of China (no. 81671483) and Beijing Municipal

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Natural Science Foundation (no. 7171011, S150002).

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Abstract word counts: 268

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Main text Word counts: 4653

28 29 30 31 32 33 1

34

Condensation and short version of title

35 36

Condensation: With multiple methods of microbiologic inquiry, no evidence was

37

found to support the presence of bacteria in the midtrimester amniotic fluid of normal

38

pregnancies.

39 40

Short Title: The absence of bacteria in midtrimester amniotic fluid

41 42

AJOG at a Glance:

43

Why was the study conducted?

44

To determine whether bacteria are present in the midtrimester amniotic fluid of

45

patients who subsequently had a normal pregnancy outcome.

46 47

Key findings

48

• Amniotic fluid did not contain any cultivable bacteria or genital mycoplasmas.

49

• There was no evidence of bacterial DNA using molecular microbiologic techniques

50

(quantitative real-time PCR and 16S rRNA gene sequencing).

51 52

What does this study add to what is already known?

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This study demonstrates that the midtrimester amniotic fluid of normal pregnancies

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does not contain bacteria using multiple methods of microbiologic inquiry.

55 56

Key words: bacteria; culture; cytokines; pregnancy outcome; 16S rRNA gene

57

sequencing; 16S rRNA gene copies

58 59 60 61 62 63 64 65 66 2

67

Abstract

68 69

BACKGROUND: There is controversy about whether the amniotic fluid contains

70

bacteria. Using sequencing-based methods, recent studies report that the amniotic

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fluid is colonized by microorganisms. However, background-contaminating DNA might

72

lead to false-positive findings when such a low microbial biomass sample is

73

examined.

74 75

OBJECTIVE: To determine whether the midtrimester amniotic fluid of patients who

76

subsequently had normal pregnancy outcomes contains a microbial signature.

77 78

STUDY DESIGN: In this prospective cohort study, 42 amniotic fluid samples were

79

collected from 37 pregnancies (5 twin and 32 singletons) during genetic

80

amniocentesis in the midtrimester. The subsequent pregnancy outcomes of all the

81

participants were followed. Multiple methods were used to detect the presence of

82

microorganisms in this study, including cultivation, quantitative real-time polymerase

83

chain reaction (qPCR) and 16S ribosomal RNA (rRNA) gene sequencing. Multiple

84

positive controls (n=16) served as quality controls, including 3 adult fecal samples, 4

85

vaginal swabs and 9 artificial bacterial communities, and run in parallel with negative

86

controls (n=12), including 4 from the hospital operating room and 8 from the laboratory,

87

to account for background-contaminating DNA during each step of the experiments.

88 89

RESULTS: (1) No bacteria under anaerobic or aerobic conditions or genital

90

mycoplasmas were cultured from any of the amniotic fluid samples. (2) Quantitative

91

PCR did not reveal greater copy numbers of 16S rRNA gene in amniotic fluid samples

92

than in negative controls. (3) 16S rRNA gene sequencing did not indicate significant

93

difference in the microbial richness or community structures between amniotic fluid

94

and negative controls.

95 96

CONCLUSION: With multiple methods of microbiologic inquiry, no microorganisms

97

were identified in the midtrimester amniotic fluid of healthy pregnancies with a normal

98

pregnancy outcome.

99 3

100 101

Introduction

102

Based on culture method, the intra-amniotic cavity has traditionally been viewed as

103

sterile for more than 100 years. However, this “sterile womb” paradigm has been

104

challenged by recent studies based on culture-independent sequencing techniques.

105

The molecular evidence of unique bacterial communities could be detected in the

106

amniotic fluid (AF),1-4 placenta,4-10 endometrium11,12 and meconium13,14 of term

107

healthy pregnancies .

108 109

Microbial invasion of the amniotic cavity (MIAC) resulting in intra-amniotic infection

110

has been associated with adverse pregnancy outcomes,15-17 such as spontaneous

111

preterm

112

histological/clinical chorioamnionitis.22,23 An ascending pathway is considered the

113

most common route of MIAC, given the direct evidence that microorganisms isolated

114

from amniotic fluid of women with intra-amniotic infection are present in the lower

115

genital tract.24

birth,15,18,19

preterm

premature

rupture

of

membranes20,21

and

116 117

Considering the unavoidable occurrence of environmental or reagent contamination

118

during the entire experimental process,25,26 multiple rigorous experimental controls

119

are urgently required when such a low microbial biomass sample is studied.27 Several

120

subsequent studies found the microbial profiles of AF28,29 and placenta30-32 to be

121

indistinguishable from negative controls, re-establishing controversy regarding the “in

122

utero colonization hypothesis”.25

123 124

As pregnancy progresses, the uterus changes from an early proinflammatory

125

condition to an antiinflammatory condition in the second trimester and then back to a

126

proinflammatory condition before the onset of labor.33 The intra-amniotic microbial

127

profile might change as the cervix shortens and dilates. Nevertheless, most studies

128

collected the samples at the time of delivery.

129 130

Using

131

polymerase chain reaction (qPCR) and16S rRNA gene sequencing, we prospectively

132

conducted a prospective cohort study to investigate the presence of microorganisms

multiple

inquiry

methods,

including

4

cultivation,

quantitative

real-time

133

in the midtrimester AF of 37 women in parallel with multiple negative and positive

134

controls and followed their subsequent pregnancy outcomes.

135

Materials and Methods

136

Study design

137

This prospective cohort study was conducted in Peking University First Hospital from

138

May 2018 to March 2019. Women undergoing amniocentesis for prenatal diagnosis

139

between 19 and 22 weeks of gestation were recruited for the study. The exclusion

140

criteria were as follows: (1) fetal malformation; (2) clinical infection and antibiotic

141

treatment within 2 weeks; and (3) refusal to participate. This study was reviewed and

142

approved by the institutional ethics committee of Peking University First Hospital

143

(2015[886]), and all the participants provided the written informed consent.

144 145

Ultimately, 37 women (5 with dichorionic diamniotic twin pregnancies and 32 with

146

singleton pregnancy) were enrolled, and 42 AF samples were collected by

147

amniocentesis (Figure 1). The clinical characteristics were obtained via electronic

148

medical records, including the history of spontaneous or in vitro fertilization (IVF)

149

conception and pregnancy outcomes (Table S1).

150 151

To reflect the potential DNA contamination during the experimental procedure, 12

152

negative controls were designed, including (1) sterile saline solution (9 mg/mL NaCl)

153

collected in the hospital operating room (n=4), serving as sampling controls; (2) DNA

154

extraction kit buffers (n=4) collected in the laboratory, without AF samples but

155

processed exactly as AF samples, serving as extraction controls; (3) PCR

156

amplification reagents (n=2) and DNA-free water (n=2) without an extraction protocol,

157

serving as amplification controls and blank controls, respectively. Moreover, 16

158

positive controls consisting of adult stool specimens (n=3), vaginal swabs (n=4), and

159

artificial bacterial communities (n=9) spiked into AF were used as experimental quality

160

controls. Artificial bacterial communities 1-7 contained various gram-positive and

161

-negative bacteria with known numbers of colony-forming units (CFUs). Artificial

162

bacterial communities 7 was serially diluted to a 10-1 dilution (artificial bacterial

163

communities 8) and 10-2 dilution (artificial bacterial communities 9) for a lowest limit of

164

detection (Table S2). Five gradients of plasmids from Escherichia coli were set up to

165

generate the qPCR standard curve. The presence of microorganisms was determined 5

166

using (1) cultivation, (2) qPCR of the 16S rRNA gene, (3) 16S rRNA gene sequencing.

167

In summary, 46 samples were subjected to cultivation, 64 samples were subjected to

168

qPCR, 68 samples were subjected to 16S rRNA gene sequencing, and 39 AF

169

samples were subjected to cytokines detection (Figure 1). The detailed information is

170

presented in Table S3.

171 172

Clinical definition and pregnancy outcomes

173

Gestational age was determined by the date of the last menstrual period and

174

confirmed by ultrasound examination. Gravidity and parity were recorded according to

175

the current admission status. Preterm birth is defined as birth occurring prior to 37

176

gestational weeks, including spontaneous and iatrogenic preterm birth. Premature

177

rupture of membranes (PROM) is defined as spontaneous rupture of membranes

178

prior to the onset of labor. Preterm PROM (PPROM) occurs before 37 gestational

179

weeks. The diagnosis of histologic chorioamnionitis is based on the presence of acute

180

inflammatory changes in the chorionic plate and/or in the chorioamniotic membrane,

181

as previously described.34,35

182 183

Sample collection and preparation

184

Amniocentesis was performed in a sterile operating room, and sample preparation

185

was carried out by the same researcher wearing sterile mask and gloves. After

186

centrifugation at 1300 × g (10 minutes, room temperature), AF supernatants were

187

collected and divided into five aliquots in a biosafety cabinet (BSC-1500IIB2-X,

188

Biobase, Shandong, China) for further analysis. Three aliquots were placed in sterile

189

tubes and transported to the laboratory for anaerobic, aerobic and genital

190

mycoplasma cultures; the remaining two aliquots were immediately stored at -80°C

191

for molecular sequencing and cytokine detection. Four negative controls were

192

collected in the same operating room following the same sampling and aliquoting

193

procedures.

194 195

Bacterial culture

196

Each aliquot of sample (2-3 mL) was injected into a BD Bactec Lytic/10 Anaerobic/F

197

Culture Vial and a BD Bactec Plus Aerobic/F Culture Vial (Becton, Dickinson and

198

Company, USA) using a sterile syringe. The entire procedure was performed in a 6

199

biological safety cabinet, and the vials were incubated in a Bactec FX Instrument

200

(Becton, Dickinson and Company, USA) at 35°C for 5 days according to the

201

manufacturer’s protocol.36

202 203

The genital mycoplasma cultivation assay (Mycoplasma IES, Autobio, Zhengzhou,

204

China) was also performed under the manufacturer’s instructions.37 In brief, 300 µL of

205

the amniotic fluid were transferred into the reconstituted medium. Afterward, 100 µL of

206

the suspension were inoculated into the wells of the strip. All the strips were incubated

207

at 35°C ~37°C for 24 h, and the appearance of a red color indicated a positive

208

reaction and microbial growth.

209 210

DNA extraction

211

DNA extraction was performed in a biological safety cabinet under the standard

212

protocol for microbial analysis with a QIAamp DNA Stool Mini kit (Qiagen, Hilden,

213

Germany). During the entire experimental process, the study personnel wore sterile

214

laboratory coats, hairnets, face masks and sleeves. DNA concentrations of the AF

215

samples and controls were measured with a Qubit 3.0 Fluorometer (Q32866, Life

216

Technologies, Carlsbad, CA), and purified DNA was stored at -20°C .

217 218

16S rRNA qPCR

219

The bacterial DNA copy number was detected by TaqMan quantitative PCR of the

220

V3-V4

221

5’-ACTCCTAYGGGRBGCASCAGT-3’;

222

5’-CCTAGCTATTACCGCGGCTGCT-3’;

223

5’-6FAMCGGCTAACTMCGTGCCAGBHQX-3’. A 20 µL reaction contained 10 µL of

224

Premix Taq (2x) Mix (Takara, Shiga, Japan), 1 µL of DNA, and 5 µmol of primer and

225

probe. Amplifications were performed with an initial denaturation at 95°C for 2 minutes,

226

followed by 40 cycles of denaturation at 94°C for 5 seconds and annealing at 60°C for

227

30 seconds. All reactions were performed using a Roche 480 Real Time PCR

228

instrument (Roche, Basel, Switzerland). A plasmid containing the 16S PCR amplicon

229

from E. coli was serially diluted from 105 copies to 10 copies to generate a standard

230

curve. Each sample was amplified in duplicate.

region

of

the

16S

rRNA

gene

using

the

following:

primer-F, primer-R, probe,

231 7

232

16S rRNA gene sequencing

233

The 16S rRNA gene V3-V4 region was chosen for Illumina sequencing to identify the

234

bacterial taxonomic composition by a two-step PCR. Extracted DNA was first

235

amplified by digital droplet PCR (ddPCR). Droplet generation, droplet transfer, and

236

plate sealing were performed according to the protocol. DNA was amplified with 1x

237

KAPA HiFi Master Mix (16SAFP02, Coyote, Beijing, China), 0.2 µmol of each primer

238

(primer-F:

239

5’-GGACTACNNGGGTATCTAAT-3’), and 9 µL of DNA. The conditions were as

240

follows: 95°C for 3 minutes, followed by 30 cycles of denaturation at 98°C for 15

241

seconds, annealing at 50°C for 50 seconds, and exte nsion at 72°C for 30 seconds,

242

with 1 cycle at 72°C for 10 minutes. All reactions were performed using a 96-well PCR

243

instrument (Coyote), and amplification products were purified with VAHTS clean

244

beads (Na44-02, Coyote). After attachment of barcode adapters (16SAFP03, Coyote),

245

the second PCR was performed under the same conditions as above, with only 8

246

cycles and an increased annealing temperature of 58°C for 30 seconds. Amplicon

247

libraries were purified with VAHTS clean beads (Na44-02, Coyote) and quantified with

248

a Qubit dsDNA HS Assay Kit (Q32851, Life Technologies). The final library was

249

sequenced using the Illumina HiSeq 2500 platform (San Diego, CA).

5’-CCTAYGGGRBGCASCAG-3’;

primer-R:

250 251

Multiplex bead array assay for cytokines

252

The AF concentrations of the following 21 cytokines were measured with an EMD

253

Millipore Milliplex Kit (HCYTO-60K, 21X-Milliplex, Billerica, MA, USA) according to the

254

manufacturer’s instructions. Thirteen proinflammatory cytokines [transforming growth

255

factor alpha (TGF-α), granulocyte colony stimulating factor (G-CSF), interferon

256

gamma (IFN-γ), interleukin-12P40 (IL-12P40), IL-15, sCD40L, IL-17A, IL-1α, IL-1β,

257

IL-2, IL-6, IL-7, and vascular endothelial growth factor (VEGF)], 3 antiinflammatory

258

cytokines [fibroblast growth factor-2 (FGF-2), IL-10, and IL-4], and 5 chemokines

259

[fractalkine, macrophage-derived chemokine (MDC), IL-8, interferon gamma-induced

260

protein 10 (IP-10), and macrophage inflammatory protein (MIP)-1α] were quantified.

261

Briefly, each well of 96-well plates was loaded in duplicate with 25 µL of assay buffer

262

and 25 µL of standard, control, or AF supernatant. Next, 25 µL of magnetic beads

263

were added into each well and incubated for 2 hours at room temperature; the wells

264

were washed twice with 200 µL of wash buffer, followed by the addition of 25 µL of 8

265

detection antibody. The plates were then incubated for 1 hour at room temperature,

266

after which 25 µL of streptavidin-phycoerythrin were added to each well and incubated

267

for 30 minutes at room temperature. The plates were washed twice more with wash

268

buffer, and 150 µL of the drive fluid were added into each well for measurement using

269

a Luminex Magpix instrument (Thermo Scientific, Waltham, MA). Standard curves

270

were generated, and the values of samples were calculated from the curve.

271 272

Statistical analysis

273

The cycle threshold (CT) values of the qPCR assay, defined as the number of thermal

274

cycles required for the detection threshold, were converted to copy numbers

275

according to the standard curve. Sequences of the 16S rRNA gene were clustered

276

using QIIME with 97% nucleotide similarity and taxonomically classified using the

277

Greengenes database. Paired reads were merged with flash software with a

278

maximum of 10% allowed between the number of mismatched base pairs and the

279

overlap length. Sequencing of DNA extracts yielded 3,884,287 sequences for AF

280

samples, 2,149,237 sequences for positive controls, and 3,366 sequences for

281

negative controls. Based on 97% nucleotide similarity, the sequences clustered into

282

400 operational taxonomic units (OTUs) for AF samples, 2,010 OTUs for positive

283

controls, and 13 OTUs for negative controls. The Good’s coverage values of all but 1

284

AF sample exceeded 99.8%. The exception was 99.7% (S18). Good’s coverage

285

values of all positive controls and negative controls exceeded 99.9%. For analyses of

286

alpha diversity, individual sample libraries were subsampled using the cutoff value of

287

30,000, and samples with fewer than 30,000 sequences were not subsampled.

288 289

Bacterial compositions were visualized with a heat map, which was generated via

290

Seaborn, a Python data visualization library. Alpha diversity was evaluated with the

291

Chao1 and Simpson indexes. Beta diversity was assessed by unweighted UniFrac

292

distance matrices and visualized by principal coordinates analysis (PCoA), with 1000

293

permutations, and statistically calculated by the nonparametric multivariate analysis of

294

variance (NPMANOVA) methods using the Adonis function in the R package vegan.

295

The metric variable was shown as the mean ± standard deviation (SD) or median

296

(interquartile range), and compared by Student’s t test or Mann-Whitney U test

297

according to the normality of the data distribution. Chi-square and Fisher’s exact tests 9

298

were used to compare of proportions of analytes. A P value < 0.05 was considered

299

significant. GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA) was

300

used for the statistical and graphical analyses.

301 302

Results

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Clinical characteristics

304

The demographic and clinical characteristics of all the patients are shown in Table 1.

305

Among the 37 women, 14 conceived with IVF (5 with dichorionic diamniotic twins) and

306

23 conceived spontaneously. One woman with twin gestation (subject ID 3) had an

307

intrauterine fetal demise of one twin at 22 weeks. Two (subject IDs 4 and 7) of four

308

cases of preterm birth had PPROM and delivered at 36 and 30 gestational weeks,

309

respectively, and the histologic acute chorioamnionitis (stage 2, grade 2) was

310

identified in subject ID 4. One woman (subject ID 10) delivered electively at 36

311

gestational weeks for gestational hypertension, and another one (subject ID 13)

312

delivered electively at 33 gestational weeks due to preeclampsia (Table S1).

313 314

Bacterial cultures

315

All the AF samples and sampling controls did not yield any bacterial cultivars under

316

aerobic or anaerobic conditions, nor the growth of genital mycoplasmas.

317 318

Real-time qPCR for 16S rRNA gene copy number

319

To quantify the microbial biomass in AF samples, we used the qPCR assay to

320

measure the copy number of the 16S rRNA gene. A standard curve over a range of 10

321

to 105 gene copies was generated by the linear regression analysis of an E. coli

322

plasmid (Figure 2A, slope= -3.7, R2 =0.99). Compared with stool samples [median

323

(min, max), 2.4x108 (2.1x108, 2.6x108) copies/µL] and vaginal swabs [4.5x107 (1.3x107,

324

7.6x107) copies/µL], AF samples contained very low bacterial biomass [553 (32,

325

24106) copies/µL]. Considering the potential contamination, we assessed the 16S

326

rRNA gene copy number in the negative controls. Although the AF samples contained

327

higher numbers of 16S gene copies than the extraction [22 (4, 287) copies/µL,

328

Mann-Whitney U test; U=9, P<0.01] and blank [14 (4, 22) copies/µL, U=0, P<0.001]

329

controls, no significant difference was observed between the AF samples and the

330

sampling controls [566 (453, 659) copies/µL, U=79.5, P=0.87] (Figure 2B). 10

331 332

16S rRNA gene sequencing

333

As the positive controls, the taxonomic composition of each artificial bacterial

334

communities was consistent with expectations (Figure 3A-G). The relative

335

abundances of bacterial compositions were highly similar among artificial bacterial

336

communities 7, 8 and 9, except for Micrococcus, which could not be accurately

337

measured in artificial bacterial communities 9 (Figure 3G), indicating the lowest

338

detection limit. Therefore, the feasibility and reliability of the sequencing data were

339

ensured. Except for 1 extraction control and 2 amplification controls, we identified

340

sequenced reads in 95.6% (65/68) of the samples. Five AF samples and 3 extraction

341

controls were removed from further analyses because no bacterial OTU was

342

annotated in these samples.

343 344

The bacterial richness of AF samples was significantly lower than that of stool (Figure

345

4A, Mann-Whitney U test, Chao1 index, U=0, P<0.001; 4B, Simpson index, U=2,

346

P<0.001). However, no significant difference was found between AF samples and

347

sampling controls (Figure 4A, Chao1 index, U=35, P=0.37; 4B, Simpson index,

348

U=36.5, P=0.42). To assess bacterial community structure, PCoA analysis based on

349

the unweighted UniFrac distance was performed. In general, the samples clustered

350

according to sample types (Figure 4C). Stool and vaginal swabs were distinct from AF

351

samples (NPMANOVA, stool vs AF: F=5.547, P<0.001; vaginal swabs vs AF: F=3.055,

352

P=0.001) and sampling controls (stool vs sampling controls: F=6.308, P=0.022;

353

vaginal swabs vs sampling controls: F=2.683, P=0.027). Similarly, no significant

354

difference in bacterial community structure was observed between the AF samples

355

and sampling controls (F=1.166, P=0.243).

356 357

To identify the potential bacterial OTUs unique to AF samples, a secondary analysis

358

was performed to eliminate the background signals in the negative controls. The

359

sequenced data were more stringently filtered (with fewer than 10 identical sequences

360

were filtered) compared to the primary analyses (with fewer than 5 identical

361

sequences were filtered). Eventually, 3 stool samples and 4 vaginal swabs, as well as

362

13 AF samples, met this threshold and possessed at least one OTU.

363 11

364

Given the absence of bacterial OTUs at the genus level, 3 AF samples (S21, S2, S10)

365

were excluded from further analysis. In the remaining 17 cases, 22 predominant

366

OTUs at the genus level were identified based on a relative abundance >1% (Figure

367

5). Among the 22 bacterial OTUs, 14 were found in AF samples, and 10 of them

368

(Pseudonocardia,

369

Bifidobacterium, Corynebacterium, Bdellovibrio and Iamia) were identified in only one

370

AF sample, suggesting that they were likely contaminants rather than genuine signals.

371

In addition, Bdellovibrio and Iamia are usually found in soil and plants and

372

Pseudonocardia and Dialister are usually found in soil,38 which are unexpected

373

findings for the human amniotic cavity from an ecological perspective. In contrast,

374

each of the remaining four bacterial OTUs, Bacteroides, Propionibacterium,

375

Faecalibacterium and Ruminococcus, was found in more than two of the AF samples.

376

which was considered ecologically plausible, as the origin might be the human vagina,

377

gut or skin. The 4 bacterial OTUs were identified in nine AF samples, including 8 (S3,

378

S4, S7, S13, S14, S16, S18 and S19) from the IVF conception and 1 (S20) from the

379

spontaneous conception (Figure 5).

Adhaeribacter,

Dialister,

Roseburia,

Delftia,

Sutterella,

380 381

Cytokine concentrations in amniotic fluid

382

To investigate any inflammatory response to the aforesaid bacterial signals, the

383

cytokine profile of AF samples was further assessed. In general, all the cytokines

384

concentrations of AF samples either with (n=7) or without (n=32) bacterial signals

385

were both considerably low, when compared with these of patients with

386

microbial-associated intra-amniotic inflammation (Table S4).39 Taking IL-6 as an

387

example, the AF concentration ≥2.6 ng/mL is used to define the intra-amniotic

388

inflammation. However, the median concentration of IL-6 of 39 AF samples was 105.5

389

pg/mL, ranged from 12.3 pg/mL to 736.9 pg/mL, which was much lower than the cutoff

390

value of intra-amniotic inflammation.

391 392

Pregnancy outcomes of women who harbored bacterial signals

393

Subsequently, we investigated the pregnancy outcomes of the women who harbored

394

the bacterial signals (Table 2). Among them, 7 women conceived through IVF and 1

395

woman conceived spontaneously. One woman (subject ID 3) had a single intrauterine

396

fetal demise at 22 weeks. Two women underwent preterm birth: one (subject ID 10) 12

397

delivered electively at 36 gestational weeks for gestational hypertension, and the

398

other (subject ID 13) delivered electively at 33 gestational weeks for preeclampsia

399

without any signs of inflammatory response during the histopathologic examination.

400

Using criteria previously described,39-41 one woman (subject ID 15) was diagnosed

401

with clinical chorioamnionitis at the time of delivery according to the presence of fever

402

(temperature was 38°C) accompanied by the symptoms of tachycardia (heart rate

403

was 106 beats/min) and leukocytosis (leukocyte count was 23,000 cells/mm3), but no

404

acute inflammatory responses were found from histopathology. No adverse

405

pregnancy outcomes were reported in remaining women.

406 407

Comment

408

Principal findings of the study

409

The main findings included (1) cultivation did not yield viable bacteria or genital

410

mycoplasmas in any AF samples; (2) qPCR did not distinguish the AF samples from

411

the negative controls based on 16S rRNA gene copy number; (3) 16S rRNA gene

412

sequencing did not reveal a difference in microbial composition or community

413

structure between AF samples and negative controls. The identification of

414

Bacteroides, Propionibacterium, Faecalibacterium and Ruminococcus against

415

negative controls, although intriguing, was not supported by the absence of

416

intra-amniotic inflammation in the cytokines detection. Overall, we did not find the

417

consistent evidence that the midtrimester AF of normal pregnancy contains

418

microorganisms.

419 420

Controversial views regarding intra-amniotic microorganisms

421

A groundbreaking publication in 20145 claimed that the placentae from uncomplicated

422

pregnancies harbor a unique microbiome that is similar to the microbiota of the human

423

oral cavity according to 16S rRNA gene sequencing, as well as metagenomics

424

sequencing in a subset of samples. This finding stimulated a wave of research

425

exploring the microbiota in placenta,4,6,10,42,43 AF,1-4 and uterus tissue,1,44,45,

426

challenging

427

accumulating studies have argued that the unavoidable background DNA

428

contamination during the experimental processes might lead to false-positive

429

findings.46-49 Recently, Rowlands et al50 found the midtrimester AF samples were

the

long-standing

“sterile

13

womb”

dogma.

However,

gradually

430

negative for the presence of bacteria, using the species-specific and broad-range

431

PCR techniques. Furthermore, Lim et al29 failed to identify a unique microbiota in AF

432

samples from healthy pregnancies that differs from negative controls. In a recent

433

study of 10 uncomplicated pregnancies with intact amniotic membranes, no significant

434

difference was observed in the bacterial loads between AF samples and negative

435

controls.28

436 437

In addition, using multiple inquiry methods and adequate technical controls, Theis et

438

al30 claimed no consistent evidence to support the existence of a unique placental

439

microbiota in patients who delivered at term without labor. More recently, de Goffau et

440

al32 also provided evidence that the placenta is not colonized by microorganisms in

441

healthy pregnancies and that any bacterial signals are associated with contamination

442

DNA and/or batch effects. Consistent with previous studies by Rowlands et al,50

443

Rehbinder et al,28 and Lim et al,29 our study included multiple technical controls and

444

multiple complementary methods of inquiry: bacterial culture, 16S rRNA gene qPCR

445

and 16S rRNA gene sequencing, and provided the robust evidence of the “sterile

446

womb”

447 448

Bacterial culture

449

Consistent with recent studies,28 no viable bacteria were cultured from any of AF

450

samples. Given that a positive culture of AF samples is the sign of MIAC,39,41 and that

451

no intra-amniotic inflammatory response was identified during the cytokines detection,

452

our study supported a sterile intra-amniotic environment at midtrimester.

453 454

qPCR

455

In line with Lim et al,29 the 16S rRNA gene copy numbers in AF were much lower than

456

those in stool samples and vaginal swabs. When compared with the negative controls,

457

the microbial abundance in AF was similar to that of the sampling controls, but higher

458

than that of the laboratory controls, indicating extra contributions of contamination

459

during the sampling process.

460 461

16S rRNA gene sequencing

462

Based on 16S rRNA gene sequencing, the bacterial richness and community 14

463

structure were similar between the negative controls and AF samples, which was

464

consistent with previous studies revealing that the amniotic cavity is sterile in

465

uncomplicated pregnancies.28,29,50 In the secondary analysis of the same data, 4

466

bacterial

467

Ruminococcus, were identified against the negative controls. As typical inhabitants in

468

the human vagina, gut or skin niches, these bacterial signals are considered to be

469

ecologically plausible. The

470

been identified in endometrium,11,12 and Propionibacterium in umbilical cord blood of

471

healthy newborn51 and chorioamnion sample of healthy pregnancies,52 suggesting the

472

possibility that these human commensal bacteria may enter the amniotic cavity

473

though ascending pathway or hematogenous dissemination.15,16

signals,

Bacteroides,

Propionibacterium,

Faecalibacterium

and

Bacteroides, Faecalibacterium and Ruminococcus have

474 475

It is noteworthy that 87.5% (7/8) of the women who harbored the four bacterial signals

476

conceived through IVF (Table 2), which may be associated with the different profiles of

477

physiology, immunology and endocrinology in the IVF pregnancies. The process of

478

IVF treatment is complicated.53 Various cytokines are involved in the balance between

479

the immunogenic resistance and tolerance.54 The exogenous estrogen and

480

progesterone in IVF treatment are associated with the risk of microbial invasion, as

481

microbes can alter and utilize host hormones to facilitate growth and survival.55 In

482

addition, IVF women are exposed to more invasive operations, such as cervical

483

excision procedures, hysteroscopic resection or induced abortion, as well as the IVF

484

treatment itself,56 which may be a route for MIAC.24 In current study, it is hard to

485

untangle the key contributors to the identification of four bacterial signals.

486 487

Cytokine profiles

488

MIAC is frequently accompanied by the presence of high AF concentration of

489

cytokines and chemokines, such as IL-1,57-59 IL-6,35,60-64 IL-8,65,66 IP-10,35,64,67 and

490

other inflammation-related proteins.68-70 The IL-6 plays a key role in diagnosis of

491

intra-amniotic inflammation, given the strong association with preterm labor and an

492

increased rate of neonatal morbidity and mortality.61,71,72 IL-1 has been implicated as a

493

signal for the onset of human parturition in the setting of infection,57 and the IL-1α

494

serves as an alarmin and plays a core role in the microbial-associated intra-amniotic

495

inflammation.72 15

496 497

Although there is a wide range of cutoffs in AF IL-6 levels for intra-amniotic

498

inflammation,61-65,71,73 any AF samples from our study did not reach the threshold

499

described in previous studies, indicating the absence of intra-amniotic inflammatory

500

response.

501

chorioamnionitis was observed in women with four bacterial signals. Therefore, no

502

further comparison was conducted. In current study, we did not find sufficient

503

evidence to support the presence of bacterial signals identified in 16S rRNA gene

504

sequencing.

Besides,

no

spontaneous

preterm

delivery

or

histopathologic

505 506

Criteria for a genuine signal

507

In combination with all previous reports, we listed the criteria of microorganisms in AF:

508

(1) absence in negative controls; (2) the 16S rRNA gene copies and microbial profiles

509

of AF are distinct from those of negative controls; (3) the presence in at least two AF

510

samples to exclude the accidental signals; (4) ecological plausibility; (5) confirmation

511

of cytokines detection; (6) using of positive controls to clarify the lowest limit of

512

detection.

513 514

Clinical implications

515

MIAC resulting in intra-amniotic inflammation or infection is a risk factor for

516

spontaneous preterm birth.17,19,74 Recent study found that the bacteria cultured from

517

amniotic fluid of women with intra-amniotic infection were identified in the vagina,

518

providing the direct evidence of ascending infection as the primary cause of

519

intra-amniotic infection.24 In our study, the MIAC is absent at midtrimester in

520

uncomplicated pregnancies. It is noteworthy that four ecologically plausible bacterial

521

signals are identified in parts of our AF samples, however, the AF cytokines

522

concentrations are far below the cutoff value for identifying the intra-amniotic

523

inflammation. As the intra-amniotic inflammation remains a key contributor to

524

spontaneous preterm delivery, our study emphasizes the necessity of AF cytokines

525

detection, especially when a possibly positive result appears.

526 527

Research implications

528

The oral cavity, vagina and gut are regarded as the high biomass sites. The amniotic 16

529

cavity and placenta, in contrast, are sterile or contain low microbial biomass. Thus,

530

the experimental conditions for testing the presence of bacteria in these tissues or

531

biological fluids need to be different from the approaches that have been used in high

532

biomass niches.27 In this study, not only negative and positive controls, but also

533

artificial bacterial communities consisting of both gram-positive and gram-negative

534

bacteria were included. Gram-positive bacteria served as quality controls, considering

535

that the high mechanical strength of the cell wall can be affected by different

536

extraction methods,75,76 The consistency between the expected and detected results

537

ensured the feasibility of the extraction method. To determine the lowest limit of

538

detection, the serial dilutions of artificial bacterial communities with known

539

concentrations were included. The detected bacterial compositions were consistent

540

with expectation, while the relative abundance at the genus level was not exactly the

541

same as that expected. Given the technical limitations of 16S rRNA gene sequencing,

542

metagenomic sequencing is warranted for further investigations if any bacterial

543

signals are found.

544 545

Strengths and limitations

546

There are several strengths of our study. First, a prospective cohort study is

547

conducted to collect AF samples at midtrimester, hence the potential impacts of

548

maternal-fetal complications and labor onset are avoided.77 Second, multiple

549

complementary modes of inquiry and two-step analysis of 16S rRNA sequence data

550

were performed to provide a more robust conclusion. Finally, the thorough negative

551

controls were included in this study, covering each step of the experiment. To ensure

552

the qualification accuracy, the artificial bacterial communities containing various

553

bacterial species with known abundances, instead of a single bacterium, were also

554

included.76

555 556

Nevertheless, the sample size is small and it is difficult to determine the origin of

557

bacterial signals found in AF samples. Furthermore, although the cytokine profile is

558

investigated in our study, the results of 16S rRNA gene sequencing were not validated

559

by metagenome sequencing.

560 561

Conclusion 17

562

With multiple methods of inquiry, we did not identify the presence of microorganisms

563

in midtrimester AF from the pregnancies with a normal pregnancy outcome.

564 565

Acknowledgments

566

We acknowledge Mr. Yichen Liu and Ms. Lanying Zhang (COYOTE Medical

567

Laboratory, Beijing, China) for contributing to the 16S rRNA gene sequencing data

568

analysis; Dr. Lingzhen Meng (Peking University First Hospital, Beijing, China) for

569

technical support on cultivation assays; Mr. Jiming Yin (Capital Medical University

570

Affiliated Beijing You An Hospital, Beijing, China) for technical support with the

571

multiplex bead array assay. The study was supported by the National Key

572

Technologies R&D Program (no. 2016YFC1000303), National Natural Science

573

Foundation of China (no. 81671483), and Beijing Municipal Natural Science

574

Foundation (no. 7171011, S150002).

575

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stain in the detection of microbial invasion in patients with preterm premature rupture

784

of membranes. Am J Obstet Gynecol. 1993;169(4):839-851.

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74. Yoon BH, Romero R, Moon JB, et al. Clinical significance of intra-amniotic

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inflammation in patients with preterm labor and intact membranes. Am J Obstet

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Gynecol. 2001;185(5):1130-1136.

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75. McOrist AL, Jackson M, Bird AR. A comparison of five methods for extraction of

789

bacterial DNA from human faecal samples. J Microbiol Methods. 2002;50(2):131-139.

790

76. Costea PI, Zeller G, Sunagawa S, et al. Towards standards for human fecal

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sample processing in metagenomic studies. Nat Biotechnol. 2017;35(11):1069-1076.

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77. Seong HS, Lee SE, Kang JH, Romero R, Yoon BH. The frequency of microbial 24

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invasion of the amniotic cavity and histologic chorioamnionitis in women at term with

794

intact membranes in the presence or absence of labor. Am J Obstet Gynecol.

795

2008;199(4):375 e371-375.

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Author and article information

808

From the Department of Obstetrics and Gynecology, Peking University First Hospital,

809

Beijing, China (Drs Liu, Tong, Qin, Feng, Wang, Wang, Ma and Yang); Beijing Key

810

Laboratory of Maternal Fetal Medicine of Gestational Diabetes Mellitus, Beijing, China

811

(Drs Liu, Tong, Qin, Feng, Wang, Wang, Ma and Yang); COYOTE Medical Laboratory,

812

Beijing, China (Ms Li, Zhao; Mr Ai); Key Laboratory of Pathogenic Microbiology and

813

Immunology/ Institute of Microbiology, Chinese Academy of Science, Beijing, China

814

(Dr Zhu)

815

The study was supported by the National Key Technologies R&D Program (no.

816

2016YFC1000303), National Natural Science Foundation of China (no. 81671483),

817

and Beijing Municipal Natural Science Foundation (no. 7171011, S150002).

818

The authors report no conflicts of interest.

819

Corresponding authors: Jingmei Ma, MD, PhD, [email protected]; Huixia Yang,

820

MD, PhD, [email protected]

821 822 823 824 825 25

826 827 828 829 830 831 832 833 834 835 836 837 838 839 840

FIGURE 1

841

The flow chart of the study design

842 843

FIGURE 2

844

Comparison of 16S rRNA gene copy number among various sample types

845

A, Standard curves of a 10-fold dilution series (105 copies to 10 copies) of Escherichia

846

coli 16S ribosomal DNA. B, Comparison of 16S rRNA gene copy numbers among

847

various sample types. Statistical significance was assessed by the Mann-Whitney U

848

test; **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, non-significant.

849

CT, cycle threshold.

850 851

FIGURE 3

852

The expected and detected compositions and relative abundances of artificial

853

bacterial communities according to 16S rRNA gene sequencing

854

A-G, the expected (left) and detected (right) bacterial relative abundance of 9 artificial

855

bacterial communities.

856 857

FIGURE 4

858

Primary analysis of 16S rRNA gene sequencing 26

859

A-B, Comparison of Chao1 (A) and Simpson (B) indexes between sample types. C,

860

Principal coordinates analysis based on unweighted UniFrac distances is shown

861

along the first two principal coordinate (PC) axes; percentages are the percent

862

variation explained by each PC axis.

863 864

FIGURE 5

865

Secondary analysis of 16S rRNA gene sequencing

866

Heat map of predominated bacterial OTUs, which have an average relative

867

abundance >1%. Asterisks indicate the bacterial OTUs is ecologically plausible.

868

IVF, in vitro fertilization.

27

TABLE 1 Clinical characteristics of the 37 study participants

Characteristics

Mean ± SD or median (IQR)

Age (years)

35.4 ± 4.4

Gravidity

2(1,2)

Parity

0(0,1)

GA at amniocentesis (weeks)

21(20,21)

GA at delivery (weeks)

39(38,39)

Birth weight (g)

3235(2743,4150)

Clinical indication, n (%) In vitro fertilization

14(37.8)

Cesarean section

24(64.9)

Preterm birth

4(10.8)

PPROM

2(5.4)

Preeclampsia

5(13.5)

Histologic chorioamnionitis

1(2.7)

SD, standard deviation; IQR, interquartile range. GA, gestational week; PPROM, preterm premature rupture of membranes.

TABLE 2 Microbiological profiles and clinical characteristics of 8 women with four bacterial signals a

Subject Predominant 16S rRNA IL-6 ID bacterial OTUs gene copies (pg/mL) 3 Bacteroides(60.8%) 3016 47.82 Faecalibacterium(8.2%) 3 Propionibacterium(30.1%) 203 42.86 Ruminococcus(23.1%) 5 Bacteroides(61.9%) 420 143.28 9 Propionibacteriu (100%) 316 N/A 10 Bacteroides(30.8%) 833 58.43

IVF/spontaneous GA at GA at conception amniocentesis delivery IVF 20 Fetal death at 22 GA IVF 20 38 IVF IVF IVF

20 21 21

11 13

Faecalibacterium(100%) 548 Ruminococcus(32.8%) 1531

102.06 132.57

IVF IVF

21 21

14 15

Bacteroides(40%) Bacteroides(100%)

N/A 144.19

IVF Spontaneous

21 21

459 4439

39 37 36 (latrogenic) 38 33 (Iatrogenic) 41 41

Pregnancy complications No

Histologic chorioamnionitis N/A

No

N/A

GDM No Gestational hypertension No Preeclampsia

N/A N/A N/A

N/A No chorioamnionitis No N/A b Clinical No chorioamnionitis chorioamnionitis

All patients (n=8) had negative cultivation results for both bacteria and mycoplasma. a

The presence of predominant bacterial OTUs among four ecologically plausible

OTUs. b

The temperature of this woman was 38°C, the heart rate was 106 beats/min, and the

leukocyte count of peripheral blood was 23,000 cells/mm3. GA, gestational age; GDM, gestational mellitus diabetes; N/A, not applicable.