Low biomass microbiota in the upper genital tract of reproductive age women: fact or fiction?,Annals of Clinical Microbiology and Antimicrobials

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Low biomass microbiota in the upper genital tract of reproductive age women: fact or fiction?
Annals of Clinical Microbiology and Antimicrobials ( IF 5.7 ) Pub Date : 2020-09-09 , DOI: 10.1186/s12941-020-00385-9
Thor Haahr _{1,

2} , Jørgen Skov Jensen ₃ , Signe Altmäe _{4,

5,

6} , Peter Humaidan _{1,

2}

Affiliation

Dear Sir,

We read with interest the recent publication by Wei et al. [1], investigating the microbiome of the reproductive tract in regular cycling women (N = 50), who underwent laparoscopic surgery for benign gynecological tumors. The study is in fact a re-analysis of a prior study [2], using a case control design to compare the microbiome of various anatomical sites along the reproductive tract; 16S rRNA gene sequencing was performed in women with verified endometriosis (staging as defined by American Fertility Association) versus controls with no endometriosis. The authors conclude that endometriosis patients have an altered endometrial and peritoneal fluid microbiota containing “signature species” as compared to non-endometriosis controls. However, before the abovementioned conclusion can be drawn, we aim to highlight important limitations which are key elements of good practice generally relevant to low biomass microbiota studies.

First, transparency and replicability are key issues—also in microbiome research [3]. Thus, the authors should be encouraged to make available the specifics of the data analysis, including the bioinformatic pipeline from raw sequences to species annotation, and also to clarify how figures were constructed. As a matter of fact, it seems that Fig. 1 only shows genus annotation and in Fig. 2 only one bacterium—Lactobacillus iners—is identified to the species level. Metadata is also lacking about the cases and controls.

Second, it is important to make available results from negative controls at all major steps through analysis. This would increase the confidence in the low abundant microbiota results being a true biological signal and not a false positive signal—e.g. from contamination. Thus, Wei et al. ought to provide microbiome results on the negative controls, e.g. a comparison in terms of sequencing depth and “signature OTUs” in the negative controls compared to samples from especially the low bio-mass sites such as the endometrium and the peritoneal fluid. In fact, more than 50% of the “signature OTUs” in Fig. 2 of the endometrial and peritoneal fluid, respectively, are well-known contaminants from sequenced blank controls [4, 5].

Third, when choosing to report new diagnostic/stratification methods, it is important to clearly state what defines them. Wei et al. stratified samples according to the dominant genus. What was the rationale behind this method? As previously mentioned, no information exists in the publication on taxon annotation. Furthermore, there is no definition as to what determines “a dominant genus”; lastly why did the authors choose seven subtypes?

Despite the somewhat unclear nature of these seven subtypes, the authors further elaborate their analysis to report the ratio between those arbitrary seven subtypes as evidence for differences between endometriosis cases and controls. Instead, we would suggest that the authors applied a rigorous analysis, utilizing the raw sequencing data, to compare alpha and beta diversity metrics etc. between endometriosis cases and controls. In their first publication [2], interesting data was reported from qPCR analyses on the total abundance of Lactobacilli ascending from the vagina to the upper reproductive tract. That information would have been interesting also in the present publication. Finally, it has been shown that ascending infection is increased in patients with bacterial vaginosis, OR 5.7 (95% CI, 1.8–18.3) [6]—could authors provide information on bacterial vaginosis status?

In summary—and in contrast to the importance statement made by the authors—we find that the data analysis itself as well as the information level regarding the data analysis in the publication are not sufficient to answer the question whether a real microbiota of the upper genital tract exists and whether it is associated with endometriosis. In our view and taking the current methods description into consideration, the conclusion of the study could also have been that upper genital tract samples were dominated by well-known contaminants which caused the observed difference between endometriosis cases and controls.

Not applicable.

1.
Wei W, Zhang X, Tang H, Zeng L, Wu R. Microbiota composition and distribution along the female reproductive tract of women with endometriosis. Ann Clin Microbiol Antimicrob. 2020;19(1):15.
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2.
Chen C, Song X, Wei W, Zhong H, Dai J, Lan Z, et al. The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases. Nat Commun. 2017;8(1):875.
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3.
Haahr T, Humaidan P, Jensen JS. Non-transparent and insufficient descriptions of non-validated microbiome methods and related reproductive outcome results should be interpreted with caution. Hum Reprod. 2019;34:2083–4.
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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(12):87.
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Eisenhofer R, Minich JJ, Marotz C, Cooper A, Knight R, Weyrich LS. Contamination in low microbial biomass microbiome studies: issues and recommendations. Trends Microbiol. 2019;27(2):105–17.
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6.
Swidsinski A, Verstraelen H, Loening-Baucke V, Swidsinski S, Mendling W, Halwani Z. Presence of a polymicrobial endometrial biofilm in patients with bacterial vaginosis. PLoS ONE. 2013;8(1):e53997.
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Affiliations

Department of Clinical Medicine, Aarhus University, Aarhus, Denmark
Thor Haahr & Peter Humaidan
The Fertility Clinic Skive, Skive Regional Hospital, Resenvej 25, 7800, Skive, Denmark
Thor Haahr & Peter Humaidan
Statens Serum Institut, Microbiology and Infection Control, Copenhagen, Denmark
Jørgen Skov Jensen
Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, 18071, Granada, Spain
Signe Altmäe
Instituto de Investigación Biosanitaria ibs.GRANADA, 18014, Granada, Spain
Signe Altmäe
Competence Centre on Health Technologies, 50410, Tartu, Estonia
Signe Altmäe

Authors

Thor HaahrView author publications
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Jørgen Skov JensenView author publications
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Signe AltmäeView author publications
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Peter HumaidanView author publications
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Contributions

TH drafted the manuscript. PH, JSJ and SA revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Thor Haahr.

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Consent for publication

All authors have read and approved this manuscript for publication.

Competing interests

TH has received honoraria for lectures from Ferring, IBSA, Besins and Merck. PH received unrestricted research grants from MSD, Merck, and Ferring as well as honoraria for lectures from MSD, Merck, Gedeon-Richter, Theramex, and IBSA. JSJ has received speaker’s fee from Hologic, BD, SpeeDx, and Cepheid and serves scientific advisory board of Roche Molecular Systems, Abbott Molecular, and Cepheid. PH, TH and JSJ received an unrestricted research grant from Osel inc. which produces LACTIN-V, a live biotherapeutic product with Lactobacillus crispatus. PH and TH are listed as inventors in an international patent application (PCT/US2018/040882) involving “Use of vaginal lactobacilli for improving the success rate of in vitro fertilization”. SA reports funding and salary from The Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526-R; FEDER/Junta de Andalucía-Consejería de Economía y Conocimiento: MENDO (B-CTS-500-UGR18).

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Haahr, T., Jensen, J.S., Altmäe, S. et al. Low biomass microbiota in the upper genital tract of reproductive age women: fact or fiction?. Ann Clin Microbiol Antimicrob 19, 41 (2020). https://doi.org/10.1186/s12941-020-00385-9

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Received: 10 June 2020
Accepted: 27 August 2020
Published: 09 September 2020
DOI: https://doi.org/10.1186/s12941-020-00385-9

中文翻译：

生殖年龄女性上生殖道中的低生物量菌群：事实还是虚构？

亲爱的先生，

我们感兴趣地阅读了Wei等人的最新出版物。[1]，研究了接受定期腹腔镜手术治疗良性妇科肿瘤的常规自行车女性（N = 50）的生殖道微生物组。该研究实际上是对先前研究的重新分析[2]，使用病例对照设计来比较生殖道各个解剖部位的微生物组。与没有子宫内膜异位的对照相比，在已证实子宫内膜异位（根据美国生育协会定义的分期）的妇女中进行16S rRNA基因测序。作者得出的结论是，与非子宫内膜异位症对照相比，子宫内膜异位症患者的子宫内膜和腹膜液微生物群发生了改变，其中含有“签名种”。但是，在得出上述结论之前，

首先，透明度和可复制性是关键问题，也是微生物组研究[3]。因此，应鼓励作者提供数据分析的细节，包括从原始序列到物种注释的生物信息流水线，并阐明数字的构造方式。实际上，图1似乎仅显示属注释，而在图2中仅在种水平上鉴定出一种细菌-乳酸杆菌。关于案例和控件的元数据也很缺乏。

其次，重要的是在分析的所有主要步骤中提供阴性对照的结果。这将增加人们对低菌群结果的信心，因为这些结果是真实的生物信号，而不是错误的阳性信号（例如来自污染）。因此，魏等。应该提供阴性对照的微生物组结果，例如，与来自特别是低生物量位点（例如子宫内膜和腹膜液）的样品相比，阴性对照的测序深度和“特征性OTU”进行比较。实际上，图2中子宫内膜和腹膜液的“特征性OTU”分别超过50％是来自已测序空白对照的众所周知的污染物[4，5]。

第三，在选择报告新的诊断/分层方法时，重要的是要清楚说明它们的定义。Wei等。根据优势属分类样品。这种方法背后的原理是什么？如前所述，出版物中没有有关分类单元注释的信息。此外，对于什么决定“显性属”没有定义。最后，为什么作者选择七个亚型？

尽管这七个亚型的性质尚不清楚，但作者进一步完善了分析，以报告这七个亚型之间的比率，以作为子宫内膜异位症病例与对照组之间差异的证据。相反，我们建议作者使用原始的测序数据进行严格的分析，以比较子宫内膜异位症病例与对照之间的α和β多样性指标等。在他们的第一个出版物[2]中，qPCR分析报告了有关乳酸杆菌总丰度的有趣数据从阴道升至上生殖道。该信息在本出版物中也将是有趣的。最后，研究表明细菌性阴道病或OR 5.7（95％CI，1.8-18.3）[6]的患者中上升感染增加了。作者能否提供有关细菌性阴道病状态的信息？

总而言之，与作者的重要性声明相反，我们发现数据分析本身以及出版物中有关数据分析的信息水平不足以回答上生殖器的真正微生物区系是否存在以及是否与子宫内膜异位症有关。我们认为并考虑到当前的方法说明，该研究的结论也可能是上生殖道样品被众所周知的污染物所控制，这些污染物导致了子宫内膜异位症病例与对照组之间的差异。

不适用。

1。
魏W，张X，唐H，曾L，吴R.子宫内膜异位症女性生殖道上的菌群组成和分布。安临床微生物抗微生物剂。2020; 19（1）：15。
CAS文章Google学术搜索
2。
Chen C，Song X，Wei W，Zhong H，Dai J，Lan Z，et al。沿女性生殖道的微生物群连续体及其与子宫相关疾病的关系。Nat Commun。2017; 8（1）：875。
文章Google学术搜索
3。
Haahr T，Humaidan P，Jensen JS。对未验证的微生物组方法和相关生殖结果的描述不透明且描述不足，应谨慎解释。嗡嗡声。2019; 34：2083–4。
CAS文章Google学术搜索
4。
Salter SJ，Cox MJ，Turek EM，Calus ST，Cookson WO，Moffatt MF等。试剂和实验室污染会严重影响基于序列的微生物组分析。BMC生物学。2014; 12（12）：87。
文章Google学术搜索
5，
Eisenhofer R，Minich JJ，Marotz C，Cooper A，Knight R，Weyrich LS。低微生物生物量微生物组研究中的污染：问题和建议。趋势微生物。2019; 27（2）：105–17。
CAS文章Google学术搜索
6。
Swidsinski A，Verstraelen H，Loening-Baucke V，Swidsinski S，Mendling W，Halwani Z.细菌性阴道病患者体内存在多种微生物子宫内膜生物膜。一等奖。2013; 8（1）：e53997。
CAS文章Google学术搜索

下载参考

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隶属关系

奥尔胡斯大学临床医学系，丹麦奥尔胡斯
托尔·哈尔（Thor Haahr）和彼得·胡迈丹（Peter Humaidan）
生育诊所Skive，Skive地区医院，Resenvej 25，7800，Skive，丹麦
托尔·哈尔（Thor Haahr）和彼得·胡迈丹（Peter Humaidan）
史坦顿血清研究所，微生物学和感染控制，丹麦哥本哈根
约尔根·斯科夫·詹森
格拉纳达大学理学院生物化学与分子生物学系，西班牙格拉纳达18071
西格·阿尔特梅
西班牙格拉纳达生物卫生研究所IBs.18014，西班牙格拉纳达
西格·阿尔特梅
爱沙尼亚塔尔图，卫生技术能力中心，50410
西格·阿尔特梅

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会费

TH起草了手稿。PH，JSJ和SA修改了手稿。所有作者阅读并认可的终稿。

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与Thor Haahr的通信。

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同意发表

所有作者均已阅读并批准该手稿以供出版。

利益争夺

TH已获得Ferring，IBSA，Besins和Merck的演讲酬金。PH收到了MSD，默克和Ferring的无限制研究资助，并获得了MSD，默克，Gedeon-Richter，Theramex和IBSA的演讲的酬金。JSJ已从Hologic，BD，SpeeDx和Cepheid收取演讲者费用，并担任Roche Molecular Systems，Abbott Molecular和Cepheid的科学顾问委员会。PH，TH和JSJ获得了Osel inc。的无限制研究资助。其产生LACTIN-V，一活生物治疗产品，卷曲乳杆菌。PH和TH被列为国际专利申请（PCT / US2018 / 040882）的发明人，该申请涉及“使用阴道乳杆菌提高体外受精的成功率”。SA报告了来自西班牙经济，工业和竞争力部（MINECO）和欧洲区域发展基金（FEDER）的资金和薪资：RYC-2016-21199和ENDORE SAF2017-87526-R；费德/安达卢西亚·孔塞伊拉·德·埃科诺米扬经济协会：门多（B-CTS-500-UGR18）。

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