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Female reproductive tract microbiota and recurrent pregnancy loss: a nested case-control study

  • Author Footnotes
    # These authors contributed equally to this work.
    Pirkko Peuranpää
    Correspondence
    Corresponding author.
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Obstetrics and Gynaecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
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  • Author Footnotes
    # These authors contributed equally to this work.
    Tiina Holster
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    # These authors contributed equally to this work.
    Affiliations
    Department of Obstetrics and Gynaecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
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  • Schahzad Saqib
    Affiliations
    Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
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  • Ilkka Kalliala
    Affiliations
    Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Department of Metabolism, Digestion, and Reproduction, Faculty of Medicine, Imperial College London, London, UK
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  • Aila Tiitinen
    Affiliations
    Department of Obstetrics and Gynaecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
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    # These authors contributed equally to this work.
    Anne Salonen
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    # These authors contributed equally to this work.
    Affiliations
    Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
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  • Author Footnotes
    # These authors contributed equally to this work.
    Hanna Hautamäki
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Obstetrics and Gynaecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
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    # These authors contributed equally to this work.
Open AccessPublished:June 16, 2022DOI:https://doi.org/10.1016/j.rbmo.2022.06.008

      Highlights

      • Recurrent pregnancy loss often remains unexplained in standard examinations
      • 16S rRNA gene amplicon sequencing is used to analyse the reproductive tract microbiota
      • The endometrium has its own microbiota, with a composition close to that of the vaginal microbiota
      • A dysbiotic reproductive tract microbiota is associated with recurrent miscarriages
      • A dysbiotic endometrial microbiota may be a novel background cause for miscarriages

      Abstract

      Research question

      Is the composition of the endometrial or vaginal microbiota associated with recurrent pregnancy loss (RPL)?

      Design

      Endometrial and vaginal samples were collected from 47 women with two or more consecutive pregnancy losses and 39 healthy control women without a history of pregnancy loss, between March 2018 and December 2020 at Helsinki University Hospital, Helsinki, Finland. The compositions of the endometrial and vaginal microbiota, analysed using 16S rRNA gene amplicon sequencing, were compared between the RPL and control women, and between individual vaginal and endometrial samples. The mycobiota composition was analysed using internal transcribed spacer 1 amplicon sequencing for a descriptive summary. The models were adjusted for body mass index, age and parity. False discovery rate-corrected P-values (q-values) were used to define nominal statistical significance at q < 0.05.

      Results

      Lactobacillus crispatus was less abundant in the endometrial samples of women with RPL compared with controls (mean relative abundance 17.2% versus 45.6%, q = 0.04). Gardnerella vaginalis was more abundant in the RPL group than in controls in both endometrial (12.4% versus 5.8%, q < 0.001) and vaginal (8.7% versus 5.7%, q = 0.002) samples. The individual vaginal and endometrial microbial compositions correlated strongly (R = 0.85, P < 0.001). Fungi were detected in 22% of the endometrial and 36% of the vaginal samples.

      Conclusions

      Dysbiosis of the reproductive tract microbiota is associated with RPL and may represent a novel risk factor for pregnancy losses.

      Key words

      Introduction

      Recurrent pregnancy loss (RPL) is one of the most challenging issues in reproductive medicine because its causes are often unknown and effective treatment is rarely available. The European Society of Human Reproduction and Embryology defines RPL as the spontaneous loss of two or more pregnancies (
      • Bender Atik R.
      • Christiansen O.B.
      • Elson J.
      • Kolte A.M.
      • Lewis S.
      • Middeldorp S.
      • Nelen W.
      • Peramo B.
      • Quenby S.
      • Vermeulen N.
      • Goddijn M.
      ESHRE guideline: recurrent pregnancy loss.
      ), and it affects 1–3% of couples trying to have a child. Recognized causes of RPL are chromosomal abnormalities, uterine malformations, antiphospholipid syndrome and endocrinological disorders (
      • Rai R.
      • Regan L.
      Recurrent miscarriage.
      ), but over half of RPL still remains unexplained.
      Several studies have suggested an infectious aetiology behind miscarriages (
      • Giakoumelou S.
      • Wheelhouse N.
      • Cuschieri K.
      • Entrican G.
      • Howie S.E.M.
      • Horne A.W.
      The role of infection in miscarriage.
      ;
      • McQueen D.B.
      • Perfetto C.O.
      • Hazard F.K.
      • Lathi R.B.
      Pregnancy outcomes in women with chronic endometritis and recurrent pregnancy loss.
      ). Chronic endometritis has been associated with RPL (
      • McQueen D.B.
      • Maniar K.P.
      • Hutchinson A.
      • Confino R.
      • Bernardi L.
      • Pavone M.E.
      Redefining chronic endometritis: the importance of endometrial stromal changes.
      ) and bacterial vaginosis has been linked to the risk of miscarriage (
      • Haahr T.
      • Zacho J.
      • Bräuner M.
      • Shathmigha K.
      • Skov Jensen J.
      • Humaidan P.
      Reproductive outcome of patients undergoing in vitro fertilisation treatment and diagnosed with bacterial vaginosis or abnormal vaginal microbiota: a systematic PRISMA review and meta-analysis.
      ;
      • Ralph S.G.
      • Rutherford A.J.
      • Wilson J.D.
      Influence of bacterial vaginosis on conception and miscarriage in the first trimester: Cohort study.
      ). Recent studies using high-throughput DNA sequencing techniques have shown that Lactobacillus spp. dominate the vaginal bacterial composition in healthy early pregnancy (
      • Freitas A.C.
      • Chaban B.
      • Bocking A.
      • Rocco M.
      • Yang S.
      • Hill J.E.
      • Money D.M.
      • Hemmingsen S.
      • Reid G.
      • Dumonceaux T.
      • Gloor G.
      • Links M.
      • O'Doherty K.
      • Tang P.
      • van Schalkwyk J.
      • Yudin M.
      The vaginal microbiome of pregnant women is less rich and diverse, with lower prevalence of Mollicutes, compared to non-pregnant women.
      ;
      • MacIntyre D.A.
      • Chandiramani M.
      • Lee Y.S.
      • Kindinger L.
      • Smith A.
      • Angelopoulos N.
      • Lehne B.
      • Arulkumaran S.
      • Brown R.
      • Teoh T.G.
      • Holmes E.
      • Nicoholson J.K.
      • Marchesi J.R.
      • Bennett P.R.
      The vaginal microbiome during pregnancy and the postpartum period in a European population.
      ), while dysbiosis, the reduced prevalence of lactobacilli, especially Lactobacillus crispatus, has been associated with pregnancy loss (
      • Al-Memar M.
      • Bobdiwala S.
      • Fourie H.
      • Mannino R.
      • Lee Y.S.
      • Smith A.
      • Marchesi J.R.
      • Timmerman D.
      • Bourne T.
      • Bennett P.R.
      • MacIntyre D.A.
      The association between vaginal bacterial composition and miscarriage: a nested case–control study.
      ). The mycobiota, consisting of various species of fungi, is another important element of the vaginal ecosystem (
      • Bradford L.L.
      • Ravel J.
      The vaginal mycobiome: A contemporary perspective on fungi in women's health and diseases.
      ). However, there are no studies examining the mycobiota in relation to reproductive outcomes.
      The uterine cavity has long been thought to be sterile, but recent studies have reported that the endometrium may have a distinct microbiome (
      • Chen C.
      • Song X.
      • Wei W.
      • Zhong H.
      • Dai J.
      • Lan Z.
      • Li F.
      • Yu X.
      • Feng Q.
      • Wang Z.
      • Xie H.
      • Chen X.
      • Zeng C.
      • Wen B.
      • Zeng L.
      • Du H.
      • Tang H.
      • Xu C.
      • Xia Y.
      • Xia H.
      • Yang H.
      • Wang Jian
      • Wang Jun
      • Madsen L.
      • Brix S.
      • Kristiansen K.
      • Xu X.
      • Li J.
      • Wu R.
      • Jia H.
      The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
      ;
      • Mitchell C.M.
      • Haick A.
      • Nkwopara E.
      • Garcia R.
      • Rendi M.
      • Agnew K.
      • Fredricks D.N.
      • Eschenbach D.
      Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
      ;
      • Moreno I.
      • Codoñer F.M.
      • Vilella F.
      • Valbuena D.
      • Martinez-Blanch J.F.
      • Jimenez-Almazán J.
      • Alonso R.
      • Alamá P.
      • Remohí J.
      • Pellicer A.
      • Ramon D.
      • Simon C.
      Evidence that the endometrial microbiota has an effect on implantation success or failure.
      ). As in the vagina, the dominant species in the endometrium are usually lactobacilli (
      • Chen C.
      • Song X.
      • Wei W.
      • Zhong H.
      • Dai J.
      • Lan Z.
      • Li F.
      • Yu X.
      • Feng Q.
      • Wang Z.
      • Xie H.
      • Chen X.
      • Zeng C.
      • Wen B.
      • Zeng L.
      • Du H.
      • Tang H.
      • Xu C.
      • Xia Y.
      • Xia H.
      • Yang H.
      • Wang Jian
      • Wang Jun
      • Madsen L.
      • Brix S.
      • Kristiansen K.
      • Xu X.
      • Li J.
      • Wu R.
      • Jia H.
      The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
      ,
      • Oberle A.
      • Urban L.
      • Falch-Leis S.
      • Ennemoser C.
      • Nagai Y.
      • Ashikawa K.
      • Ulm P.A.
      • Hengstschläger M.
      • Feichtinger M.
      16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.
      ), and an alteration of this composition may affect reproductive outcomes. In patients undergoing IVF, a non-Lactobacillus dominated endometrial microbiota has been associated with lower implantation rates, clinical pregnancy rates and live birth rates compared with a Lactobacillus-dominated microbiota (
      • Moreno I.
      • Codoñer F.M.
      • Vilella F.
      • Valbuena D.
      • Martinez-Blanch J.F.
      • Jimenez-Almazán J.
      • Alonso R.
      • Alamá P.
      • Remohí J.
      • Pellicer A.
      • Ramon D.
      • Simon C.
      Evidence that the endometrial microbiota has an effect on implantation success or failure.
      ). However, knowledge of the endometrial microbiota in RPL is scarce.
      The goal of this study was to explore the composition of the microbiota and mycobiota in endometrial and vaginal samples in women with RPL and compare the results with those of healthy women without a history of miscarriages. The study also investigated whether the composition of the vaginal microbiota reflected the composition of the endometrial microbiota.

      Material and methods

      Study population

      TOIVE is a prospective cohort study of the immunological and microbiological causes of RPL, conducted at the University of Helsinki and Helsinki University Hospital (HUS), Finland. Between March 2018 and June 2020, the study recruited 51 women referred to the Reproductive Medicine Unit and the Department of Obstetrics and Gynaecology of Hyvinkää Hospital because of RPL (Figure 1). Women were eligible if they had a history of three or more consecutive clinical first-trimester pregnancy losses or two losses with at least one in the second trimester, and had no concomitant infertility. Clinical pregnancy loss was defined as a spontaneous loss of an intrauterine pregnancy confirmed by ultrasonography or a positive urine or serum human chorionic gonadotrophin test, and over 6 weeks of amenorrhoea.
      Figure 1
      Figure 1Flow chart describing the study design. RPL, recurrent pregnancy loss.
      As controls, 40 women investigated for male factor infertility between June 2018 and December 2020 were recruited. They represented healthy Finnish women as they had no history of pregnancy loss, endometriosis, anovulation or Fallopian tube defects. Participants were excluded if they were <18 or ≥40 years of age, had hepatitis or HIV infection, or had an irregular menstrual cycle (<21 or >42 days).
      Power calculations were not applicable to this associative and novel setting as earlier studies on microbiota composition and RPL were lacking. The ethics committee of HUS approved the study (no. HUS/3635/2017; date of approval 17 January 2018).

      Collection of clinical data

      All participants gave their written informed consent. Upon enrolment, a detailed medical and reproductive history was taken, and participants received a questionnaire related to previous infections, the use of anti- or probiotics, sexual behaviour and educational background. The basic examinations for RPL included transvaginal 2D-ultrasonography, a complete blood count, phospholipid antibodies, karyotyping (of both partners), thyroid function tests, thyroid peroxidase antibodies, fasting glucose and glycated haemoglobin. Screening for thrombophilia, coeliac disease, hyperprolactinaemia and uterine anomalies with 3D-ultrasonography or hysteroscopy was performed when necessary. Women were not routinely tested for sexually transmitted diseases or chronic endometritis.

      Endometrial and vaginal sampling

      Vaginal and endometrial samples were collected in the mid-luteal phase, 6–8 days after a positive ovulation test (Clearblue Digital; Swiss Precision Diagnostics, Switzerland). Couples were advised to use a condom or abstain from sexual intercourse during the menstrual cycle in which the samples were taken, to ensure contraception and to avoid the effects of seminal fluid on the microbiota. Samples from the control women were collected during a natural menstrual cycle preceding the woman's first IVF/intracytoplasmic sperm injection stimulation; women with RPL were not undergoing active fertility treatment.
      Vaginal samples were collected during a speculum examination from the right and left fornices with sterile flocked swabs (FLOQSwabs; Copan, Italy) and severed to 1.5 ml Eppendorf tubes. The researchers used non-sterile examination gloves and a white coat during sampling, but not a mask or hair cover. Lubricants were not used. Endometrial samples were collected in 1.5 ml Eppendorf tubes using an endometrial biopsy curette (Pipelle; Prodimed, France), inserted gently through the cervix into the uterine cavity, without contact with the vaginal walls but unprotected when passing through the cervical canal. In the uterine cavity, a vacuum was created by retracting the internal piston, and the sample was collected by rotating the device and gently moving it back and forth. The Eppendorf tubes were frozen at −20°C immediately after sampling, and moved to −80 °C within 2 weeks.

      DNA extraction

      Microbial DNA was extracted from the vaginal samples using a beat beating method as previously described (
      • Virtanen S.
      • Rantsi T.
      • Virtanen A.
      • Kervinen K.
      • Nieminen P.
      • Kalliala I.
      • Salonen A.
      Vaginal Microbiota Composition Correlates Between Pap Smear Microscopy and Next Generation Sequencing and Associates to Socioeconomic Status.
      ). One-sixth of the endometrial biopsies (average weight 33 mg; SD 22 mg) were used for DNA extraction, with negative controls using the same method as for the vaginal samples. DNA was quantified using a Quanti-iT PicoGreen dsDNA Assay (Invitrogen, USA). The DNA yields for both sample types were comparable, with a mean of 78.8 ng/µl (SD 60 ng/µl) for the vaginal and 43.8 ng/µl (SD 30 ng/µl) for the endometrial samples. The negative controls (no input sample for DNA extraction) did not contain detectable amounts of DNA. Nine samples from an earlier project were resequenced as positive controls.

      16S rRNA gene and internal transcribed spacer (ITS) amplicon sequencing

      MiSeq (Illumina , USA) paired-end sequencing of PCR amplicons from the hypervariable V3–V4 regions of the bacterial 16S rRNA gene (primers 341F/785R) and fungal ITS-1 region (primers ITS1F and ITS2) was prepared and performed as explained in detail elsewhere (

      Virtanen, S., Saqib, S., Kanerva, T., Nieminen, P., Kalliala, I., Salonen, A., 2021. Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiota and Mycobiota in Healthy Women.https://www.researchsquare.com/article/rs-321778/v1

      ). Briefly, bacterial and fungal amplicons were prepared separately and combined for indexing in a 1:1 ratio, and an equimolar pool was sequenced using 2  ×  300 bp reads and a MiSeq v3 reagent kit at the Biomedicum Functional Genomics Unit, Helsinki, Finland.

      Sequence processing and analysing

      The primary step of the analysis was to split the combined 16S rRNA gene and ITS sequence FASTQ files into separate datasets. This was done by first removing ambiguous (N) bases from the reads, followed by primer-based separation using cutadapt v3.5, removing the primers in the process (
      • Martin M.
      Cutadapt removes adapter sequences from high-throughput sequencing reads.
      ). From this step onwards, all pre-processing was carried out individually on both datasets.
      The bacterial 16S rRNA gene dataset was processed using dada2 v1.20, following the pipeline tutorial v1.16, while the ITS pipeline tutorial v1.8 was used for processing the ITS sequence dataset (
      • Callahan B.J.
      • McMurdie P.J.
      • Rosen M.J.
      • Han A.W.
      • Johnson A.J.A.
      • Holmes S.P.
      DADA2: High-resolution sample inference from Illumina amplicon data.
      ). The dada2 tutorials were followed only until the stage of amplicon sequence variant (ASV) table construction and removal of chimaeras, after which the taxminer package was used to assign taxonomic annotations (

      Saqib, S., 2021. Taxonomic annotations – BLAST alignment and text-mining based filtration in R [WWW Document].https://github.com/SchahzadSaqib/taxminer.

      ). This is a Basic Local Alignment Search Tool (BLAST)-based annotation tool combined with text-mining-based filtration to assign the most likely annotations. A detailed description of this approach has been reported elsewhere (

      Virtanen, S., Saqib, S., Kanerva, T., Nieminen, P., Kalliala, I., Salonen, A., 2021. Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiota and Mycobiota in Healthy Women.https://www.researchsquare.com/article/rs-321778/v1

      ).
      Briefly, for sequence alignment, a stringent threshold of 98% was set for both percentage identity and query coverage, and was supplemented the authors’ taxonomic filtration approach, which extracts the host and isolation source of each sequence alignment hit. The former eliminates low-quality alignments, and the latter filters the results based on user-defined parameters, in this case Homo sapiens (host) and female reproductive tract/clinical isolates/gut (isolation source), effectively minimizing the presence of potential contaminants and misannotations within the results.
      The taxonomic profiles of vaginal bacterial communities can be sorted into categories called community state types (CST), a classification method based on the dominance, depletion or absence of prominent vaginal bacteria, mainly lactobacilli, within the bacterial profiles. CST were assigned in both vaginal and endometrial samples using the VALENCIA method (
      • France M.T.
      • Ma B.
      • Gajer P.
      • Brown S.
      • Humphrys M.S.
      • Holm J.B.
      • Waetjen L.E.
      • Brotman R.M.
      • Ravel J.
      VALENCIA: a nearest centroid classification method for vaginal microbial communities based on composition.
      ).

      Statistical analysis

      Statistical analyses were performed using IBM SPSS Statistics, version 25 (IBM Corporation, USA) and R (version 4.1.0; The R Foundation, Austria). A two-sample t-test and Mann–Whitney U-test were used to compare the continuous background variables, and Pearson chi-squared and Fisher's exact tests to compare the categorical variables between the RPL and control groups. Parity was categorized as nulliparous and parous (≥1 delivery).
      The primary outcomes were the mean relative abundances of bacteria and fungi in the endometrial and vaginal samples. Associations between background variables and the microbiota were analysed using permutational analysis of variance (PERMANOVA) for the overall microbiota variation using the adonis2 function with 99,999 permutations from the vegan package (

      Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., Minchin, P.R., O'Hara, R.B., Simpson, G.L., Solymos, P., Stevens, M.H.H., Szoecs, E., Wagner, H., 2020. Vegan: Community Ecology Package. R package version 2.5-7 [WWW Document].https://CRAN.R-project.org/package=vegan.

      ). Potential confounders to adjust models for differential abundance analysis downstream were identified based on the following criteria: (i) factors previously identified to influence microbiota and RPL; and (ii) confounders with statistical significance, determined by PERMANOVA analysis, ordination plots and the GroupTest function from the package mare (

      Korpela, K., 2016. Mare: Microbiota Analysis in R Easily. R package version 1.0 [WWW Document].https://github.com/katrikorpela/mare.

      ). GroupTest performs differential abundance analysis for taxa within user-defined groups, in this case each background variable. The function finds the most suitable model for individual taxa and uses read counts as an offset within the model formula to account for sequencing depth. Finally, cases and controls were used as the grouping variable in GroupTest to determine the significantly different taxa in endometrial and vaginal samples between the RPL and control groups. Based on the criteria mentioned above, body mass index (BMI), parity and age were selected as confounders. The P-values obtained were adjusted for multiple testing by false discovery rate correction and reported as q-values to define nominal statistical significance at q < 0.05. The correlations between an individual's vaginal and endometrial microbial profiles were investigated using Pearson's correlation coefficient.

      Results

      Samples were taken from 47 women with RPL and 39 control women to study the composition of the endometrial and vaginal microbiota and mycobiota. In one woman with RPL, only a vaginal sample was obtained. The participants with RPL had a history of three (range 2–5) consecutive miscarriages, of which 13 (15.3%) had occurred during the second trimester of pregnancy. Nine women had a likely explanation for their miscarriages: five had been diagnosed with congenital uterine malformation, three with acquired thrombophilia, and one with antiphospholipid syndrome and chromosomal translocation.
      The women with RPL were older (mean 33.2 [range 22–39] versus 32.1 [26–38] years, P = 0.04) and had a higher BMI (mean 25.4 [19.5–39.4] versus 23.3 [19.4–33.1] kg/m2, P = 0.02), and were more often parous (50.0% versus 15.4%, P < 0.001) than the controls (Table 1). The RPL group more often had self-reported recurrent (≥3) vaginal candidiasis, bacterial vaginosis and regular oral or vaginal probiotic use than the control group.
      TABLE 1  
      VariableRPL group (n = 46)Control group (n = 39)P-value
      Age, years0.04
       Mean (SD [range])33.2 (3.9 [22–39])32.1 (3.0 [26–38])
      BMI, kg/m20.02
       Mean (SD [range])25.4 (4.2 [19.5–39.4])23.3 (3.1 [19.4–33.1])
      Parity, n (%)<0.001
       Nulliparous (n = 56)23 (50.0)33 (84.6)
       Parous (n = 29)23 (50.0)6 (15.4)
      Prior curettage, n (%)<0.001
       Yes (n = 18)18 (39.1)0
       No (n = 67)28 (60.9)39 (100.0)
      Folic acid use, n (%)
      Data missing in four cases.
      0.02
       Yes (n = 71)34 (79.1)37 (97.4)
       No (n = 10)9 (20.9)1 (2.6)
      Vitamin D use, n (%)
      Data missing in four cases.
      0.21
       Yes (n = 70)35 (81.4)35 (92.1)
       No (n = 11)8 (18.6)3 (7.9)
      Iron use, n (%)
      Data missing in four cases.
      0.34
       Yes (n = 30)18 (41.9)12 (31.6)
       No (n = 51)25 (58.1)26 (68.4)
      Smoking status, n (%)0.19
       Current or former smoker (n = 16)11 (23.9)5 (12.8)
       Non-smoker (n = 69)35 (76.1)34 (87.2)
      Alcohol use, n (%)
      Data missing in three cases.
      0.11
       Yes (n = 60)29 (65.9)31 (81.6)
       No (n = 22)15 (34.1)7 (18.4)
      Prior chlamydia, gonorrhoea or genital herpes, n (%)
      Data missing in five cases.
      0.49
       Yes (n = 9)6 (14.3)3 (7.9)
       No (n = 71)36 (85.7)35 (92.1)
      Prior vaginal candidiasis, n (%)
      Data missing in five cases.
      0.45
       Yes (n = 52)29 (69.0)23 (60.5)
       No (n = 28)13 (31.0)15 (39.5)
      Recurrent vaginal candidiasis (≥3), n (%)
      Data missing in five cases.
      0.006
       Yes (n = 8)8 (19.0)0
       No (n = 72)34 (81.0)38 (100.0)
      Prior bacterial vaginosis, n (%)
      Data missing in five cases.
      0.15
       Yes (n = 16)11 (26.2)5 (13.2)
       No (n = 64)31 (73.8)33 (86.8)
      Recurrent bacterial vaginosis (≥3), n (%)
      Data missing in five cases.
      0.01
       Yes (n = 7)7 (16.7)0
       No (n = 73)35 (83.3)38 (100.0)
      Antibiotic use during the past 3 months, n (%)
      Data missing in five cases.
      0.05
       Yes (n = 11)9 (21.4)2 (5.3)
       No (n = 69)33 (78.6)36 (94.7)
      Use of probiotics, n (%)
      Data missing in six cases. BMI, body mass index; RPL, recurrent pregnancy loss.
      0.008
       Daily or weekly (n = 19)15 (36.6)4 (10.5)
       Less than weekly or never (n = 60)26 (63.4)34 (89.5)
      Level of education, n (%)
      Data missing in six cases. BMI, body mass index; RPL, recurrent pregnancy loss.
      0.75
       Low (comprehensive or vocational secondary school) (n = 20)11 (26.8)9 (23.7)
       High (upper secondary school, university) (n = 59)30 (73.2)29 (76.3)
      Missing data were excluded from the analyses.
      a Data missing in four cases.
      b Data missing in three cases.
      c Data missing in five cases.
      d Data missing in six cases.BMI, body mass index; RPL, recurrent pregnancy loss.

      16S rRNA gene sequencing results

      After taxonomic annotations and quality filtration (>500 reads), 74% (34/46) of the RPL and 77% (30/39) of control women's endometrial samples remained for bacterial analysis, with a mean read count of approximately 4500 (535–20,829), and approximately 4500 (569–11,540), respectively. Similarly, 98% of the vaginal RPL samples (46/47) and 100% of the controls (39/39) remained, with an average read count of approximately 23,600 (3728–47,923) and approximately 24,300 (1322–55,011).
      PERMANOVA analysis showed a difference in the overall composition of the endometrial microbiota between the RPL and control women (R2 (effect size) = 0.050, P = 0.01; see supplemental material Figure S1, Table S1), but the vaginal bacterial compositions were similar between the groups (Figure S2, Table S1). BMI was a strong clinical explanatory factor for the endometrial microbiota (R2 = 0.057, P = 0.005), especially among the women with RPL (R2 = 0.09, P = 0.005). Current vaginitis symptoms, use of vitamin D and folic acid, microbiota diversity and read count explained the variability of the vaginal microbiota in the whole study population, as did BMI, age and probiotics among the RPL group. Parity, gravidity or curettage had no impact on the endometrial or vaginal microbiota variation.
      For the endometrial samples, 37 bacterial species were identified (Figure 2). Lactobacillus crispatus was significantly less abundant and L. jensenii more abundant in the RPL group compared with the controls (mean relative abundance of L. crispatus 17.2% versus 45.6%, q = 0.04; L. jensenii 5.6% versus 3.6%, q = 0.004) (Figure 3, Table S2). Lactobacillus iners was the dominant endometrial bacterium in the RPL group (mean relative abundance 32.2% in the RPL group, 20.0% in the controls). Gardnerella vaginalis was more abundant in the RPL group compared with the control group (12.4% versus 5.8%, q < 0.001). None of the intestinal bacteria, such as Escherichia coli, Blautia spp. and Faecalibacterium spp., or the uncultured bacteria (bacteria that lack culture-based genomic and physiological characterization) were significantly differentially abundant in the women with RPL compared with the control participants.
      Figure 2
      Figure 2Stacked bar plots showing bacterial relative abundances in women with recurrent pregnancy loss (RPL) and control women, ordered based on the top three most prevalent and abundant taxa (Lactobacillus crispatus, Lactobacillus iners and Gardnerella vaginalis). (A) Endometrial samples, and (B) vaginal samples.
      Figure 3
      Figure 3Illustration and summary of the main results and statistical analysis. (A, B) Bacterial mean relative abundances within the endometrial/vaginal and recurrent pregnancy loss (RPL)/control subgroups. (C–E) Violin-boxpots showing the distribution of data in each subgroup for taxa that were significantly differentially abundant after adjusting for age, parity and body mass index in the endometrial (C), vaginal (D) and pooled (E) samples. False discovery rate-corrected P-values (q-values) <0.05 indicate statistically significant differences between the RPL and control (reference) subgroups. ‘Pooled’ refers to the combined results for the endometrial and vaginal samples.
      For the vaginal samples (Figure 2), the RPL group had more G. vaginalis (mean relative abundance 8.7% in the RPL group versus 5.7% in the control group, q = 0.002) and less Garderella leopoldii (1.0% versus 3.2%, respectively, q < 0.001), whereas L. crispatus was the most abundant bacterium (35.1% in the RPL group versus 47.5% in the controls) (Figure 3, Table S2). In the pooled endometrial and vaginal samples, L. crispatus was less abundant and G. vaginalis more abundant in the women with RPL than the controls (L. crispatus 27.5% versus 46.6%, q = 0.04; G. vaginalis 10.2% versus 5.7%, q < 0.001). When women with another explanation for RPL (congenital uterine malformation, acquired thrombophilia or chromosomal translocation) were excluded from the analyses, the differences in endometrial L. crispatus and endometrial and vaginal G. vaginalis abundances between the RPL and control women remained (Figure S3).
      The results of the VALENCIA analyses (Figure S4, Table S3) were in line with the species-specific results presented above. In the endometrium, CST I, characterized by L. crispatus dominance, was most frequently assigned to controls (56.7%, 17/30), while its prevalence in women with RPL was only 23.6% (8/34) (Figure S4, Table S4). Meanwhile, assignments to CST III, characterized by L. iners dominance, were observed in 44.1% (15/34) of the RPL group and 26.7% (8/30) of the controls. For non-Lactobacillus-dominated CST of interest, G. vaginalis-rich CST IV-B (14.7%, 5/34) and CST IV-C (5.9%, 2/34), characterized by heterogenous bacteria such as Streptococcus, Bifidobacterium and Prevotella spp., were detected in the RPL group and CST IV-B (6.7%, 2/30) in control participants. In the vagina, CST I was most frequently assigned to both the RPL (34.8%, 16/46) and control (48.7%, 19/39) groups, while assignment to CST III was nearly proportional (30.4%, 14/46; 30.8%, 12/39). CST IV-B and CST IV-C were marginally more frequently assigned to the RPL group (13.0%, 6/46; 6.5%, 3/46) than the control group (5.1%, 2/39; 2.6 1/39).
      There were three blank samples within this pipeline that were analysed with the same pre-processing and filtration criteria. Only two out of these three samples had a considerable number of reads, which totalled to approximately 8500 (Figure S5A). These annotated to Streptococcus oralis (around 4000), Fannyhessia vaginae (around 600), Prevotella amnii (around 1700) and Sneathia vaginalis (around 1300). Streptococcus oralis, a potential oral contaminant, was not detected in any other samples. Fannyhessia vaginae, P. amnii and S. vaginae are known vaginal microbes, and their presence in blank samples is most likely due to cross-contamination from the primary study samples. Due to these reasons, these reads were not subtracted from the sample data. Furthermore, none of these bacteria were significant in the downstream analysis, and were therefore not crucial for the main study comparisons. Nine positive controls were used within this sequencing run (Figure S5B), which had been independently sequenced, processed and annotated for an earlier project. The microbial profiles obtained from the two runs were nearly identical (Pearson's correlation coefficient 0.996).

      ITS sequencing results

      After taxonomic annotations and quality filtration, 24% (11/46) of the RPL group's and 21% (8/39) of the control group's endometrial samples remained for the fungal analysis, with an average read count of approximately 14,500 (1031–81,809) and approximately 12,000 (1383–23,999) respectively. Similarly, 36% (17/47) of the RPL and 36% (14/39) of the control women's vaginal samples remained after filtration, with an average read count of around 6000 (742–22,500) and around 13,000 (542–30,685). As fungi could only be detected in a small fraction of the samples, they may not sufficiently represent the entire study cohort. The authors therefore refrain from statistical analysis and present a descriptive overview of the taxonomic profiles.
      The most prevalent genus was Candida, which was detected in 19/39 samples (Figure S6). Candida albicans was not detected in the endometrium of women with RPL, although it was the most abundant taxon in their vaginal samples (mean relative abundance 26.9%) and in the endometrial and vaginal samples of the control group (mean relative abundance 49.9% and 42.9%, respectively). On the other hand, Candida parapsilosis was detected only in the RPL group, with an average relative abundance of 18.2% in endometrial and 11.8% in vaginal samples.

      Comparison of microbiota in vaginal and endometrial samples

      To study the relationship between the microbiota colonizing the endometrium and vagina, a comparison was made between 63 paired samples collected from these anatomical sites in the same woman. There was a strong within-individual correlation between the composition of the vaginal and endometrial microbiota (mean Pearson's correlation coefficient 0.85, P < 0.001; Figure 4, Figures S7 and S8), and 90.5% (57/63) of these sample pairs were assigned to the same CST, while 71.4% (45/63) were assigned to the same sub-CST, illustrating a high overlap in their bacterial profiles (Figure S4, Table S4). This overlap was the strongest for the Lactobacillus-dominated samples, while intestinal and uncultured bacteria were more abundant in the endometrium, especially in women with RPL.
      Figure 4
      Figure 4(A) Polar stacked bar plot illustrating the similarity of taxonomic profiles between the endometrial (right) and vagina (left) samples. The samples are arranged in the same order from top to bottom, creating a mirror image between the two semi-circles and allowing a direct comparison between the sample types. (B) Pearson's correlation between the paired endometrial and vaginal samples, arranged in the same order.

      Discussion

      An association was observed between RPL and reduced L. crispatus and increased G. vaginalis abundances in the endometrium, and increased G. vaginalis abundance in the vagina. The composition of vaginal microbiota was in concordance with that of the endometrial microbiota.
      The finding of a reduced abundance of L. crispatus in RPL endometrial samples is in line with prior studies reporting that, in the vagina, L. crispatus dominance is associated with a healthy microbial environment (
      • Kindinger L.M.
      • Bennett P.R.
      • Lee Y.S.
      • Marchesi J.R.
      • Smith A.
      • Cacciatore S.
      • Holmes E.
      • Nicholson J.K.
      • Teoh T.G.
      • MacIntyre D.A.
      The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk.
      ;
      • Petrova M.I.
      • Reid G.
      • Vaneechoutte M.
      • Lebeer S.
      Lactobacillus iners: Friend or Foe?.
      ). Furthermore, L. crispatus has been shown to be less abundant in the endometrium of women with chronic endometritis (
      • Liu Y.
      • Ko E.Y.L.
      • Wong K.K.W.
      • Chen X.
      • Cheung W.C.
      • Law T.S.M.
      • Chung J.P.W.
      • Tsui S.K.W.
      • Li T.C.
      • Chim S.S.C.
      Endometrial microbiota in infertile women with and without chronic endometritis as diagnosed using a quantitative and reference range-based method.
      ), a condition associated with RPL (
      • McQueen D.B.
      • Maniar K.P.
      • Hutchinson A.
      • Confino R.
      • Bernardi L.
      • Pavone M.E.
      Redefining chronic endometritis: the importance of endometrial stromal changes.
      ). In contrast, L. iners, which was the most dominant microbe in the endometrium of the RPL group, has been associated with dysbiosis (
      • Petrova M.I.
      • Reid G.
      • Vaneechoutte M.
      • Lebeer S.
      Lactobacillus iners: Friend or Foe?.
      ) and adverse reproductive outcomes, including subfertility (
      • Campisciano G.
      • Iebba V.
      • Zito G.
      • Luppi S.
      • Martinelli M.
      • Fischer L.
      • de Seta F.
      • Basile G.
      • Ricci G.
      • Comar M.
      Lactobacillus iners andgasseri,Prevotella bivia and hpv belong to the microbiological signature negatively affecting human reproduction.
      ), spontaneous miscarriage (
      • Nasioudis D.
      • Forney L.J.
      • Schneider G.M.
      • Gliniewicz K.
      • France M.
      • Boester A.
      • Sawai M.
      • Scholl J.
      • Witkin S.S.
      Influence of Pregnancy History on the Vaginal Microbiome of Pregnant Women in their First Trimester.
      ) and preterm birth (
      • Kindinger L.M.
      • Bennett P.R.
      • Lee Y.S.
      • Marchesi J.R.
      • Smith A.
      • Cacciatore S.
      • Holmes E.
      • Nicholson J.K.
      • Teoh T.G.
      • MacIntyre D.A.
      The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk.
      ).
      The results also showed an association between endometrial and vaginal G. vaginalis colonization and RPL. G. vaginalis is typically dominant in bacterial vaginosis, which has been associated with early (
      • Garcia-Grau I.
      • Perez-Villaroya D.
      • Bau D.
      • Gonzalez-Monfort M.
      • Vilella F.
      • Moreno I.
      • Simon C.
      Taxonomical and functional assessment of the endometrial microbiota in a context of recurrent reproductive failure: A case report.
      ;
      • Haahr T.
      • Zacho J.
      • Bräuner M.
      • Shathmigha K.
      • Skov Jensen J.
      • Humaidan P.
      Reproductive outcome of patients undergoing in vitro fertilisation treatment and diagnosed with bacterial vaginosis or abnormal vaginal microbiota: a systematic PRISMA review and meta-analysis.
      ; Moreno at al., 2016;
      • Ralph S.G.
      • Rutherford A.J.
      • Wilson J.D.
      Influence of bacterial vaginosis on conception and miscarriage in the first trimester: Cohort study.
      ) and especially with late (
      • Leitich H.
      • Kiss H.
      Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome.
      ) miscarriages. Miscarriage has also been linked with Lactobacillus depletion and high bacterial diversity in vaginal samples collected during early pregnancy (
      • Al-Memar M.
      • Bobdiwala S.
      • Fourie H.
      • Mannino R.
      • Lee Y.S.
      • Smith A.
      • Marchesi J.R.
      • Timmerman D.
      • Bourne T.
      • Bennett P.R.
      • MacIntyre D.A.
      The association between vaginal bacterial composition and miscarriage: a nested case–control study.
      ). Kuon and colleagues (
      • Kuon R.J.
      • Togawa R.
      • Vomstein K.
      • Weber M.
      • Goeggl T.
      • Strowitzki T.
      • Markert U.R.
      • Zimmermann S.
      • Daniel V.
      • Dalpke A.H.
      • Toth B.
      Higher prevalence of colonization withGardnerella vaginalis and gram-negative anaerobes in patients with recurrent miscarriage and elevated peripheral natural killer cells.
      ) reported that women with RPL who had vaginal G. vaginalis colonization showed higher peripheral blood natural killer cell levels, suggesting a link between dysbiotic reproductive tract microbiota, inflammation and miscarriage. Furthermore, Lactobacillus depletion and the presence of pathogenic bacteria such as Bifidobacterium, Gardnerella, Klebsiella and Neisseria in endometrial biopsies has been associated with unsuccessful reproductive outcomes in IVF treatment (
      • Moreno I.
      • Garcia-Grau I.
      • Perez-Villaroya D.
      • Gonzalez-Monfort M.
      • Bahçeci M.
      • Barrionuevo M.J.
      • Taguchi S.
      • Puente E.
      • Dimattina M.
      • Lim M.W.
      • Meneghini G.
      • Aubuchon M.
      • Leondires M.
      • Izquierdo A.
      • Perez-Olgiati M.
      • Chavez A.
      • Seethram K.
      • Bau D.
      • Gomez C.
      • Valbuena D.
      • Vilella F.
      • Simon C.
      Endometrial microbiota composition is associated with reproductive outcome in infertile patients.
      ). In the current study, women with RPL reported a history of bacterial vaginosis, vaginal candidiasis and the use of probiotics more often than controls. Although bacterial vaginosis and vaginal candidiasis were not clinically or microscopically verified, symptoms and the use of probiotics may reflect these women's susceptibility to vaginal infection.
      The genital tract microbiota is dynamic, influenced by several factors, including ethnicity (
      • Fettweis J.M.
      • Paul Brooks J.
      • Serrano M.G.
      • Sheth N.U.
      • Girerd P.H.
      • Edwards D.J.
      • Strauss J.F.
      • Jefferson K.K.
      • Buck G.A.
      Differences in vaginal microbiome in African American women versus women of European ancestry.
      ), age (
      • Wang J.
      • Li Z.
      • Ma X.
      • Du L.
      • Jia Z.
      • Cui X.
      • Yu L.
      • Yang J.
      • Xiao L.
      • Zhang B.
      • Fan H.
      • Zhao F.
      Translocation of vaginal microbiota is involved in impairment and protection of uterine health.
      ), BMI (
      • Allen N.G.
      • Edupuganti L.
      • Edwards D.J.
      • Jimenez N.R.
      • Buck G.A.
      • Jefferson K.K.
      • Strauss J.F.
      • Wickham E.P.
      • Fettweis J.M.
      The vaginal microbiome in women of reproductive age with healthy weight versus overweight/obesity.
      ), pregnancy (
      • Romero R.
      • Hassan S.S.
      • Gajer P.
      • Tarca A.L.
      • Fadrosh D.W.
      • Nikita L.
      • Galuppi M.
      • Lamont R.F.
      • Chaemsaithong P.
      • Miranda J.
      • Chaiworapongsa T.
      • Ravel J.
      The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women.
      ) and childbirth (
      • Jie Z.
      • Chen C.
      • Hao L.
      • Li F.
      • Song L.
      • Zhang X.
      • Zhu J.
      • Tian L.
      • Tong X.
      • Cai K.
      • Zhang Z.
      • Ju Y.
      • Yu X.
      • Li Y.
      • Zhou H.
      • Lu H.
      • Qiu X.
      • Li Q.
      • Liao Y.
      • Zhou D.
      • Lian H.
      • Zuo Y.
      • Chen X.
      • Rao W.
      • Ren Y.
      • Wang Y.
      • Zi J.
      • Wang R.
      • Liu N.
      • Wu J.
      • Zhang W.
      • Liu X.
      • Zong Y.
      • Liu W.
      • Xiao L.
      • Hou Y.
      • Xu X.
      • Yang H.
      • Wang J.
      • Kristiansen K.
      • Jia H.
      Life History Recorded in the Vagino-cervical Microbiome Along with Multi-omics.
      ). The vaginal microbiota is significantly different in overweight women versus women with a normal weight, demonstrating higher diversity and lower Lactobacillus dominance (
      • Allen N.G.
      • Edupuganti L.
      • Edwards D.J.
      • Jimenez N.R.
      • Buck G.A.
      • Jefferson K.K.
      • Strauss J.F.
      • Wickham E.P.
      • Fettweis J.M.
      The vaginal microbiome in women of reproductive age with healthy weight versus overweight/obesity.
      ). As women with RPL had a higher BMI than the control group in the current study population, the results were adjusted by BMI to eliminate its potential effect on the findings. Endometrial and vaginal microbiota alter as women age (
      • Wang J.
      • Li Z.
      • Ma X.
      • Du L.
      • Jia Z.
      • Cui X.
      • Yu L.
      • Yang J.
      • Xiao L.
      • Zhang B.
      • Fan H.
      • Zhao F.
      Translocation of vaginal microbiota is involved in impairment and protection of uterine health.
      ). The most significant changes occur after the age of 50 years, while the microbiota seems to be rather stable between ages 20 and 40 (
      • Wang J.
      • Li Z.
      • Ma X.
      • Du L.
      • Jia Z.
      • Cui X.
      • Yu L.
      • Yang J.
      • Xiao L.
      • Zhang B.
      • Fan H.
      • Zhao F.
      Translocation of vaginal microbiota is involved in impairment and protection of uterine health.
      ). Although age seems not to affect the reproductive tract microbiota for the ages of the current study population, it was selected as a confounder because age is strongly associated with RPL (
      • Lund M.
      • Kamper-Jørgensen M.
      • Nielsen H.S.
      • Lidegaard Ø.
      • Andersen A.M.N.
      • Christiansen O.B.
      Prognosis for live birth in women with recurrent miscarriage: What is the best measure of success?.
      ), it differed between the RPL and control groups, and it explained the variability in endometrial and vaginal microbiota in PERMANOVA. Because the control group had had significantly fewer childbirths than the RPL group, and the lack of previous childbirths has been associated with cervicovaginal L. crispatus colonization (
      • Jie Z.
      • Chen C.
      • Hao L.
      • Li F.
      • Song L.
      • Zhang X.
      • Zhu J.
      • Tian L.
      • Tong X.
      • Cai K.
      • Zhang Z.
      • Ju Y.
      • Yu X.
      • Li Y.
      • Zhou H.
      • Lu H.
      • Qiu X.
      • Li Q.
      • Liao Y.
      • Zhou D.
      • Lian H.
      • Zuo Y.
      • Chen X.
      • Rao W.
      • Ren Y.
      • Wang Y.
      • Zi J.
      • Wang R.
      • Liu N.
      • Wu J.
      • Zhang W.
      • Liu X.
      • Zong Y.
      • Liu W.
      • Xiao L.
      • Hou Y.
      • Xu X.
      • Yang H.
      • Wang J.
      • Kristiansen K.
      • Jia H.
      Life History Recorded in the Vagino-cervical Microbiome Along with Multi-omics.
      ), the results were also adjusted for parity.
      Lactobacillus-dominated endometrial microbiota may support early pregnancy, while more diverse microbiota can be detrimental, although the underlying mechanisms are still poorly understood (
      • Al-Nasiry S.
      • Ambrosino E.
      • Schlaepfer M.
      • Morré S.A.
      • Wieten L.
      • Voncken J.W.
      • Spinelli M.
      • Mueller M.
      • Kramer B.W.
      The Interplay Between Reproductive Tract Microbiota and Immunological System in Human Reproduction.
      ;
      • Bardos J.
      • Fiorentino D.
      • Longman R.E.
      • Paidas M.
      Immunological Role of the Maternal Uterine Microbiome in Pregnancy: Pregnancies Pathologies and Alterated Microbiota.
      ;
      • Benner M.
      • Ferwerda G.
      • Joosten I.
      • van der Molen R.G.
      How uterine microbiota might be responsible for a receptive, fertile endometrium.
      ). Liu and co-workers (
      • Liu F.T.
      • Yang S.
      • Yang Z.
      • Zhou P.
      • Peng T.
      • Yin J.
      • Ye Z.
      • Shan H.
      • Yu Y.
      • Li R.
      An Altered Microbiota in the Lower and Upper Female Reproductive Tract of Women with Recurrent Spontaneous Abortion.
      ) observed higher bacterial richness and diversity with altered cytokine concentrations in the endometrial fluid samples of women with RPL compared with control women. In vitro, L. crispatus has been shown to attach to the decidualized endometrial cells and prevent pathogenetic microbes occupying the attachment sites (
      • Shiroda M.
      • Manning S.D.
      Lactobacillus strains vary in their ability to interact with human endometrial stromal cells.
      ). Conversely, dysbiotic endometrial microbiota may weaken the epithelial tight junctions, allowing pathogens to enter the endometrial stroma and induce a harmful immune reaction (
      • Al-Nasiry S.
      • Ambrosino E.
      • Schlaepfer M.
      • Morré S.A.
      • Wieten L.
      • Voncken J.W.
      • Spinelli M.
      • Mueller M.
      • Kramer B.W.
      The Interplay Between Reproductive Tract Microbiota and Immunological System in Human Reproduction.
      ). The activation of Toll-like receptors on the surface of endometrial cells by microbial molecules may elicit the secretion of cytokines that alter the local immune environment (
      • Benner M.
      • Ferwerda G.
      • Joosten I.
      • van der Molen R.G.
      How uterine microbiota might be responsible for a receptive, fertile endometrium.
      ), leading to poor natural killer cell maturation. This may provoke disturbances in placentation (
      • Al-Nasiry S.
      • Ambrosino E.
      • Schlaepfer M.
      • Morré S.A.
      • Wieten L.
      • Voncken J.W.
      • Spinelli M.
      • Mueller M.
      • Kramer B.W.
      The Interplay Between Reproductive Tract Microbiota and Immunological System in Human Reproduction.
      ) as natural killer cells are essential in trophoblast invasion (
      • Moffett A.
      • Shreeve N.
      First do no harm: Uterine natural killer (NK) cells in assisted reproduction.
      ) and remodelling of the spiral arteries (
      • Smith S.D.
      • Dunk C.E.
      • Aplin J.D.
      • Harris L.K.
      • Jones R.L.
      Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.
      ). Abnormal endometrial microbiota may also favour endometrial T-helper 1 cell types (
      • Bardos J.
      • Fiorentino D.
      • Longman R.E.
      • Paidas M.
      Immunological Role of the Maternal Uterine Microbiome in Pregnancy: Pregnancies Pathologies and Alterated Microbiota.
      ), which is thought to predispose to RPL (
      • Wang W.
      • Sung N.
      • Gilman-Sachs A.
      • Kwak-Kim J.
      T Helper (Th) Cell Profiles in Pregnancy and Recurrent Pregnancy Losses: Th1/Th2/Th9/Th17/Th22/Tfh Cells.
      ).
      The origin of endometrial microbes is still unclear, but the vagina and the gastrointestinal tract have been suggested (
      • Bardos J.
      • Fiorentino D.
      • Longman R.E.
      • Paidas M.
      Immunological Role of the Maternal Uterine Microbiome in Pregnancy: Pregnancies Pathologies and Alterated Microbiota.
      ). The current findings and those of other researchers (
      • Walther-António M.R.S.
      • Chen J.
      • Multinu F.
      • Hokenstad A.
      • Distad T.J.
      • Cheek E.H.
      • Keeney G.L.
      • Creedon D.J.
      • Nelson H.
      • Mariani A.
      • Chia N.
      Potential contribution of the uterine microbiome in the development of endometrial cancer.
      ;
      • Wang J.
      • Li Z.
      • Ma X.
      • Du L.
      • Jia Z.
      • Cui X.
      • Yu L.
      • Yang J.
      • Xiao L.
      • Zhang B.
      • Fan H.
      • Zhao F.
      Translocation of vaginal microbiota is involved in impairment and protection of uterine health.
      ) of a concordance between vaginal and endometrial microbiota speak in favour of the vaginal route. Other possible mechanisms include haematogenous spread from the gastrointestinal tract or retrograde ascension from the peritoneal cavity through the Fallopian tubes. Interestingly, oxygen-sensitive intestinal bacteria, including Blautia and Faecalibacterium spp., were detected in endometrial samples. In addition, Verstraelen and collaborators (
      • Verstraelen H.
      • Vilchez-Vargas R.
      • Desimpel F.
      • Jauregui R.
      • Vankeirsbilck N.
      • Weyers S.
      • Verhelst R.
      • Sutter P.de
      • Pieper D.H.
      • de Wiele T.van
      Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.
      ) detected Bacteroides spp. in the endometrial samples of women with RPL or recurrent implantation failure, which supports the theory of a peritoneal route. However, although intestinal bacteria were detected solely in the endometrial samples in the current study, it cannot be excluded that these bacteria have ascended from the perineum through the vagina and remained under the detection level due to unfavourable conditions, such as a low pH. It is unlikely that the intestinal bacteria found in the endometrial samples were reagent-derived contaminants as those are typically water- and soil-associated bacterial genera (
      • Salter S.J.
      • Cox M.J.
      • Turek E.M.
      • Calus S.T.
      • Cookson W.O.
      • Moffatt M.F.
      • Turner P.
      • Parkhill J.
      • Loman N.J.
      • Walker A.W.
      Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
      ) that were either not detected or were disregarded in the final sample read counts. In addition, intestinal bacteria were not found within the negative control samples. Finally, the role of intestinal versus vaginal microbes in the uterus and their potential effects on reproductive health remain to be elucidated.
      This study has several strengths and limitations. The study population was ethnically and even genetically homogenous. Sampling was timed to the mid-secretory phase to analyse the microbiota during the receptive state of the endometrium, as the composition of the vaginal (
      • Lopes dos Santos Santiago G.
      • Tency I.
      • Verstraelen H.
      • Verhelst R.
      • Trog M.
      • Temmerman M.
      • Vancoillie L.
      • Decat E.
      • Cools P.
      • Vaneechoutte M.
      Longitudinal qPCR Study of the Dynamics ofL. crispatus, L. iners, A. vaginae, (Sialidase Positive)G. vaginalis, andP. bivia in the Vagina.
      ) and endometrial (
      • Kadogami D.
      • Nakaoka Y.
      • Morimoto Y.
      Use of a vaginal probiotic suppository and antibiotics to influence the composition of the endometrial microbiota.
      ;
      • Khan K.N.
      • Fujishita A.
      • Masumoto H.
      • Muto H.
      • Kitajima M.
      • Masuzaki H.
      • Kitawaki J.
      Molecular detection of intrauterine microbial colonization in women with endometriosis.
      ) microbiota may vary throughout the menstrual cycle. The recommended technique of transcervical biopsy was used for collecting endometrial tissue (
      • Molina N.M.
      • Sola-Leyva A.
      • Haahr T.
      • Aghajanova L.
      • Laudanski P.
      • Castilla J.A.
      • Altmäe S.
      Analysing endometrial microbiome: Methodological considerations and recommendations for good practice.
      ). In addition to the microbiota, the endometrial mycobiota were also analysed, which has not previously been explored. The control group in this study represent the general population as closely as possible as they were healthy and did not have any previous miscarriages or conditions known to be associated with alterations in reproductive tract microbiota, such as endometriosis (
      • Khan K.N.
      • Fujishita A.
      • Masumoto H.
      • Muto H.
      • Kitajima M.
      • Masuzaki H.
      • Kitawaki J.
      Molecular detection of intrauterine microbial colonization in women with endometriosis.
      ;
      • Wei W.
      • Zhang X.
      • Tang H.
      • Zeng L.
      • Wu R.
      Microbiota composition and distribution along the female reproductive tract of women with endometriosis.
      ), endometrial polyps (
      • Fang R.L.
      • Chen L.X.
      • Shu W.S.
      • Yao S.Z.
      • Wang S.W.
      • Chen Y.Q.
      Barcoded sequencing reveals diverse intrauterine microbiomes in patients suffering with endometrial polyps.
      ), polycystic ovary syndrome (
      • Tu Y.
      • Zheng G.
      • Ding G.
      • Wu Y.
      • Xi J.
      • Ge Y.
      • Gu H.
      • Wang Y.
      • Sheng J.
      • Liu X.
      • Jin L.
      • Huang H.
      Comparative Analysis of Lower Genital Tract Microbiome Between PCOS and Healthy Women.
      ) or Fallopian tube occlusion (
      • Haahr T.
      • Zacho J.
      • Bräuner M.
      • Shathmigha K.
      • Skov Jensen J.
      • Humaidan P.
      Reproductive outcome of patients undergoing in vitro fertilisation treatment and diagnosed with bacterial vaginosis or abnormal vaginal microbiota: a systematic PRISMA review and meta-analysis.
      ). However, the applicability of these results to the general population remains to be explored.
      As a limitation, low microbial abundance specimens, such as the endometrium, are susceptible to contamination (
      • O'Callaghan J.L.
      • Turner R.
      • Dekker Nitert M.
      • Barrett H.L.
      • Clifton V.
      • Pelzer E.S.
      Re-assessing microbiomes in the low-biomass reproductive niche.
      ). Although the authors avoided contacting the vaginal walls during sampling, cervicovaginal contamination cannot be ruled out as the back-and-forth movement of the biopsy device can inevitably push cervical mucus into the uterine cavity and contaminate the endometrial sample, and similarities existed between individual vaginal and endometrial microbial profiles. However, growing evidence supports the theory that the vaginal and endometrial ecosystems are not separate but can share microbes (
      • Chen C.
      • Song X.
      • Wei W.
      • Zhong H.
      • Dai J.
      • Lan Z.
      • Li F.
      • Yu X.
      • Feng Q.
      • Wang Z.
      • Xie H.
      • Chen X.
      • Zeng C.
      • Wen B.
      • Zeng L.
      • Du H.
      • Tang H.
      • Xu C.
      • Xia Y.
      • Xia H.
      • Yang H.
      • Wang Jian
      • Wang Jun
      • Madsen L.
      • Brix S.
      • Kristiansen K.
      • Xu X.
      • Li J.
      • Wu R.
      • Jia H.
      The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
      ;
      • Walther-António M.R.S.
      • Chen J.
      • Multinu F.
      • Hokenstad A.
      • Distad T.J.
      • Cheek E.H.
      • Keeney G.L.
      • Creedon D.J.
      • Nelson H.
      • Mariani A.
      • Chia N.
      Potential contribution of the uterine microbiome in the development of endometrial cancer.
      ;
      • Wang J.
      • Li Z.
      • Ma X.
      • Du L.
      • Jia Z.
      • Cui X.
      • Yu L.
      • Yang J.
      • Xiao L.
      • Zhang B.
      • Fan H.
      • Zhao F.
      Translocation of vaginal microbiota is involved in impairment and protection of uterine health.
      ), and only a minority of studies have questioned the existence of a uterine microbiota (
      • Winters A.D.
      • Romero R.
      • Gervasi M.T.
      • Gomez-Lopez N.
      • Tran M.R.
      • Garcia-Flores V.
      • Pacora P.
      • Jung E.
      • Hassan S.S.
      • Hsu C.D.
      • Theis K.R.
      Does the endometrial cavity have a molecular microbial signature?.
      ). Although cervical mucus protects the uterine environment, spermatozoa pass from the vagina to the uterus, and vaginally administered radioactively labelled albumin macrospheres spread in the uterine cavity within minutes (
      • Kunz G.
      • Beil D.
      • Deiniger H.
      • Einspanier A.
      • Mall G.
      • Leyendecker G.
      The uterine peristaltic pump: Normal and impeded sperm transport within the female genital tract.
      ). Therefore, it is likely that microbes ascending from the vagina may colonize the endometrium. In addition, the composition of the endometrial microbiota has been reported to be highly similar in laparoscopically and transcervically taken samples (
      • Chen C.
      • Song X.
      • Wei W.
      • Zhong H.
      • Dai J.
      • Lan Z.
      • Li F.
      • Yu X.
      • Feng Q.
      • Wang Z.
      • Xie H.
      • Chen X.
      • Zeng C.
      • Wen B.
      • Zeng L.
      • Du H.
      • Tang H.
      • Xu C.
      • Xia Y.
      • Xia H.
      • Yang H.
      • Wang Jian
      • Wang Jun
      • Madsen L.
      • Brix S.
      • Kristiansen K.
      • Xu X.
      • Li J.
      • Wu R.
      • Jia H.
      The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
      ), and common vaginal microbes, including lactobacilli, have been found in the endometrium even after hysterectomy (
      • Chen C.
      • Song X.
      • Wei W.
      • Zhong H.
      • Dai J.
      • Lan Z.
      • Li F.
      • Yu X.
      • Feng Q.
      • Wang Z.
      • Xie H.
      • Chen X.
      • Zeng C.
      • Wen B.
      • Zeng L.
      • Du H.
      • Tang H.
      • Xu C.
      • Xia Y.
      • Xia H.
      • Yang H.
      • Wang Jian
      • Wang Jun
      • Madsen L.
      • Brix S.
      • Kristiansen K.
      • Xu X.
      • Li J.
      • Wu R.
      • Jia H.
      The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
      ;
      • Miles S.M.
      • Hardy B.L.
      • Merrell D.S.
      Investigation of the microbiota of the reproductive tract in women undergoing a total hysterectomy and bilateral salpingo-oopherectomy.
      ;
      • Mitchell C.M.
      • Haick A.
      • Nkwopara E.
      • Garcia R.
      • Rendi M.
      • Agnew K.
      • Fredricks D.N.
      • Eschenbach D.
      Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
      ). Overall, even if there were cervicovaginal contamination of the endometrial samples, this would not explain the observed differences between the RPL and control groups.
      Environmental, reagent and cross-contamination are a major concern in every low microbial biomass microbiome study (
      • Eisenhofer R.
      • Minich J.J.
      • Marotz C.
      • Cooper A.
      • Knight R.
      • Weyrich L.S.
      Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations.
      ; Salter, 2014). Several steps were taken at various stages of this project to identify and eliminate potential contaminants from the results. Environmental or reagent contamination was not likely as bacteria found in the negative controls, such as S. oralis, were not found in the women's samples, while reads for Lactobacillus and Gardnerella species were not substantial. The presence of vaginal microbes such as F. vaginae, P. amnii and S. vaginae could be explained as contamination, but their low abundance and prevalence in the study samples, as well as their lack of significance within the group comparisons, indicates that they did not have a significant effect on the overall results.

      Conclusions

      RPL was associated with a dysbiotic female reproductive tract microbiota, especially in the uterus. A divergent endometrial microbial environment may be a new background cause for RPL, possibly contributing to an adverse immunological response during implantation and placentation. Further research should examine the mechanisms of how altered microbiota may contribute to RPL, evaluate whether the prognosis of subsequent pregnancies could be assessed according to the microbiota profile, and investigate whether the endometrial microbiota could be modified to increase the success of future pregnancies in some couples affected by RPL.

      Acknowledgements

      The researchers are deeply grateful to the women who participated in this study. They also wish to thank midwives Anneli Porkka, Sanna Erkkilä and Karoliina Hyttinen for their excellent work in caring for the women and in the schedules, laboratory coordinator Tinja Kanerva for processing the samples, and MD, MSc (Tech) Seppo Virtanen for his contribution to the study design.

      Funding

      The European Union's Horizon 2020 research and innovation programme (H2020 MSCA Sweet Crosstalk project under grant agreement No. 814102) and the Department of Obstetrics and Gynaecology, Helsinki University Hospital (No. TYH2018232) funded the study. Funds from HUS Hyvinkää Hospital (No. M3080TUT21) and the Finnish Cultural Foundation supported P.P., from the Academy of Finland went to I.K. and from the Juhani Aho Foundation for Medical Research supported P.P. and H.H.

      Data availability

      The sequencing data that support the findings of this study are available in the European Nucleotide Archive (PRJEB48310). All code and the clinical metadata have been deposited to GitHub: https://github.com/SchahzadSaqib/TOIVE.

      Appendix. Supplementary materials

      • Figure S3. Illustration and summary of the main results and statistical analysis excluding samples with explained recurrent pregnancy loss (RPL). (A-B) Bacterial mean relative abundances within the endometrial/vaginal and RPL/control subgroups. (C - E) Violin-Boxplots showing the distribution of data in each subgroup for taxa that were significantly differentially abundant after adjusting with age, parity, and BMI in the endometrial (C), vaginal (D) and pooled samples (E). False discovery rate -corrected P-values (q-values) < 0.05 indicate statistically significant differences between the RPL and control (reference) subgroups. Pooled means the combined results of endometrial and vaginal samples.

      • Figure S4. Heatmap of sample-wise relative abundances. The columns are annotated with community state types (CSTs) and subCSTs assigned with the VALENCIA classifier. Hierarchal clustering was performed using the “ward.D2” method.

      • Figure S5. The microbial profiles of control samples used within the sequencing run. A) Blank samples. The text within each bar represents the read count of the specified taxon B) Positive controls. The microbial profiles are compared between the same samples sequenced in the current and previous sequencing run. The text within the bar represents the read counts of the sample.

      • Figure S7. PCoA plots with weighted unifrac distances. A) The plots are split by sample type (endometrium or vagina) and coloured by the clinical phenotype (RPL or control). B) Vice versa.

      • Table S1. Adonis (PERMANOVA) summaries for clinical and technical variables explaining the overall microbiota variation in the endometrial and vaginal samples in women with recurrent pregnancy loss (RPL), controls, and all women.

      • Table S2. Full model summaries with age, BMI, and parity used as confounding factors. The raw P-values, False Discovery Rate (FDR) -corrected P-values (q-values), and the model chosen to test each taxon are reported. The subheadings represent the subset of data used for the tests.

      References

        • Allen N.G.
        • Edupuganti L.
        • Edwards D.J.
        • Jimenez N.R.
        • Buck G.A.
        • Jefferson K.K.
        • Strauss J.F.
        • Wickham E.P.
        • Fettweis J.M.
        The vaginal microbiome in women of reproductive age with healthy weight versus overweight/obesity.
        Obesity. 2022; 30https://doi.org/10.1002/oby.23306
        • Al-Memar M.
        • Bobdiwala S.
        • Fourie H.
        • Mannino R.
        • Lee Y.S.
        • Smith A.
        • Marchesi J.R.
        • Timmerman D.
        • Bourne T.
        • Bennett P.R.
        • MacIntyre D.A.
        The association between vaginal bacterial composition and miscarriage: a nested case–control study.
        BJOG. 2020; 127: 264-274
        • Al-Nasiry S.
        • Ambrosino E.
        • Schlaepfer M.
        • Morré S.A.
        • Wieten L.
        • Voncken J.W.
        • Spinelli M.
        • Mueller M.
        • Kramer B.W.
        The Interplay Between Reproductive Tract Microbiota and Immunological System in Human Reproduction.
        Front. Immunol. 2020; 11: 378
        • Bardos J.
        • Fiorentino D.
        • Longman R.E.
        • Paidas M.
        Immunological Role of the Maternal Uterine Microbiome in Pregnancy: Pregnancies Pathologies and Alterated Microbiota.
        Front. Immunol. 2020; 10: 2823
        • Bender Atik R.
        • Christiansen O.B.
        • Elson J.
        • Kolte A.M.
        • Lewis S.
        • Middeldorp S.
        • Nelen W.
        • Peramo B.
        • Quenby S.
        • Vermeulen N.
        • Goddijn M.
        ESHRE guideline: recurrent pregnancy loss.
        Hum. Reprod. Open. 2018; 2018: hoy004
        • Benner M.
        • Ferwerda G.
        • Joosten I.
        • van der Molen R.G.
        How uterine microbiota might be responsible for a receptive, fertile endometrium.
        Hum. Reprod. Update. 2018; 24: 393-415
        • Bradford L.L.
        • Ravel J.
        The vaginal mycobiome: A contemporary perspective on fungi in women's health and diseases.
        Virulence. 2017; 8: 342-351
        • Callahan B.J.
        • McMurdie P.J.
        • Rosen M.J.
        • Han A.W.
        • Johnson A.J.A.
        • Holmes S.P.
        DADA2: High-resolution sample inference from Illumina amplicon data.
        Nature Methods. 2016; 13https://doi.org/10.1038/nmeth.3869
        • Campisciano G.
        • Iebba V.
        • Zito G.
        • Luppi S.
        • Martinelli M.
        • Fischer L.
        • de Seta F.
        • Basile G.
        • Ricci G.
        • Comar M.
        Lactobacillus iners andgasseri,Prevotella bivia and hpv belong to the microbiological signature negatively affecting human reproduction.
        Microorganisms. 2021; 9: 10.3390
        • Chen C.
        • Song X.
        • Wei W.
        • Zhong H.
        • Dai J.
        • Lan Z.
        • Li F.
        • Yu X.
        • Feng Q.
        • Wang Z.
        • Xie H.
        • Chen X.
        • Zeng C.
        • Wen B.
        • Zeng L.
        • Du H.
        • Tang H.
        • Xu C.
        • Xia Y.
        • Xia H.
        • Yang H.
        • Wang Jian
        • Wang Jun
        • Madsen L.
        • Brix S.
        • Kristiansen K.
        • Xu X.
        • Li J.
        • Wu R.
        • Jia H.
        The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases.
        Nat. Commun. 2017; 8: 875
        • Eisenhofer R.
        • Minich J.J.
        • Marotz C.
        • Cooper A.
        • Knight R.
        • Weyrich L.S.
        Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations.
        Trends in Microbiology. 2019; https://doi.org/10.1016/j.tim.2018.11.003
        • Fang R.L.
        • Chen L.X.
        • Shu W.S.
        • Yao S.Z.
        • Wang S.W.
        • Chen Y.Q.
        Barcoded sequencing reveals diverse intrauterine microbiomes in patients suffering with endometrial polyps.
        Am. J. Transl. Res. 2016; 8: 1581-1592
        • Fettweis J.M.
        • Paul Brooks J.
        • Serrano M.G.
        • Sheth N.U.
        • Girerd P.H.
        • Edwards D.J.
        • Strauss J.F.
        • Jefferson K.K.
        • Buck G.A.
        Differences in vaginal microbiome in African American women versus women of European ancestry.
        Microbiology (United Kingdom). 2014; 160https://doi.org/10.1099/mic.0.081034-0
        • France M.T.
        • Ma B.
        • Gajer P.
        • Brown S.
        • Humphrys M.S.
        • Holm J.B.
        • Waetjen L.E.
        • Brotman R.M.
        • Ravel J.
        VALENCIA: a nearest centroid classification method for vaginal microbial communities based on composition.
        Microbiome. 2020; 8https://doi.org/10.1186/s40168-020-00934-6
        • Freitas A.C.
        • Chaban B.
        • Bocking A.
        • Rocco M.
        • Yang S.
        • Hill J.E.
        • Money D.M.
        • Hemmingsen S.
        • Reid G.
        • Dumonceaux T.
        • Gloor G.
        • Links M.
        • O'Doherty K.
        • Tang P.
        • van Schalkwyk J.
        • Yudin M.
        The vaginal microbiome of pregnant women is less rich and diverse, with lower prevalence of Mollicutes, compared to non-pregnant women.
        Sci. Rep. 2017; 7: 9212-9219
        • Garcia-Grau I.
        • Perez-Villaroya D.
        • Bau D.
        • Gonzalez-Monfort M.
        • Vilella F.
        • Moreno I.
        • Simon C.
        Taxonomical and functional assessment of the endometrial microbiota in a context of recurrent reproductive failure: A case report.
        Pathogens. 2019; 8https://doi.org/10.3390/pathogens8040205
        • Giakoumelou S.
        • Wheelhouse N.
        • Cuschieri K.
        • Entrican G.
        • Howie S.E.M.
        • Horne A.W.
        The role of infection in miscarriage.
        Hum. Reprod. Update. 2016; 22: 116-133
        • Haahr T.
        • Zacho J.
        • Bräuner M.
        • Shathmigha K.
        • Skov Jensen J.
        • Humaidan P.
        Reproductive outcome of patients undergoing in vitro fertilisation treatment and diagnosed with bacterial vaginosis or abnormal vaginal microbiota: a systematic PRISMA review and meta-analysis.
        BJOG. 2019; 126: 200-207
        • Jie Z.
        • Chen C.
        • Hao L.
        • Li F.
        • Song L.
        • Zhang X.
        • Zhu J.
        • Tian L.
        • Tong X.
        • Cai K.
        • Zhang Z.
        • Ju Y.
        • Yu X.
        • Li Y.
        • Zhou H.
        • Lu H.
        • Qiu X.
        • Li Q.
        • Liao Y.
        • Zhou D.
        • Lian H.
        • Zuo Y.
        • Chen X.
        • Rao W.
        • Ren Y.
        • Wang Y.
        • Zi J.
        • Wang R.
        • Liu N.
        • Wu J.
        • Zhang W.
        • Liu X.
        • Zong Y.
        • Liu W.
        • Xiao L.
        • Hou Y.
        • Xu X.
        • Yang H.
        • Wang J.
        • Kristiansen K.
        • Jia H.
        Life History Recorded in the Vagino-cervical Microbiome Along with Multi-omics.
        Genomics, Proteomics & Bioinformatics. 2021; https://doi.org/10.1016/j.gpb.2021.01.005
        • Kadogami D.
        • Nakaoka Y.
        • Morimoto Y.
        Use of a vaginal probiotic suppository and antibiotics to influence the composition of the endometrial microbiota.
        Reprod. Biol. 2020; 20: 307-314
        • Khan K.N.
        • Fujishita A.
        • Masumoto H.
        • Muto H.
        • Kitajima M.
        • Masuzaki H.
        • Kitawaki J.
        Molecular detection of intrauterine microbial colonization in women with endometriosis.
        Eur. J. Obstet. Gynecol. Reprod Biol. 2016; 199: 69-75
        • Kindinger L.M.
        • Bennett P.R.
        • Lee Y.S.
        • Marchesi J.R.
        • Smith A.
        • Cacciatore S.
        • Holmes E.
        • Nicholson J.K.
        • Teoh T.G.
        • MacIntyre D.A.
        The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk.
        Microbiome. 2017; 5: 6-9
      1. Korpela, K., 2016. Mare: Microbiota Analysis in R Easily. R package version 1.0 [WWW Document].https://github.com/katrikorpela/mare.

        • Kunz G.
        • Beil D.
        • Deiniger H.
        • Einspanier A.
        • Mall G.
        • Leyendecker G.
        The uterine peristaltic pump: Normal and impeded sperm transport within the female genital tract.
        Adv. Exp. Med. Biol. 1997; 424: 267-277
        • Kuon R.J.
        • Togawa R.
        • Vomstein K.
        • Weber M.
        • Goeggl T.
        • Strowitzki T.
        • Markert U.R.
        • Zimmermann S.
        • Daniel V.
        • Dalpke A.H.
        • Toth B.
        Higher prevalence of colonization withGardnerella vaginalis and gram-negative anaerobes in patients with recurrent miscarriage and elevated peripheral natural killer cells.
        J. Reprod. Immunol. 2017; 120: 15-19
        • Leitich H.
        • Kiss H.
        Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome.
        Best Pract. Res. Clin. Obstet. Gynaecol. 2007; 21: 375-390
        • Liu F.T.
        • Yang S.
        • Yang Z.
        • Zhou P.
        • Peng T.
        • Yin J.
        • Ye Z.
        • Shan H.
        • Yu Y.
        • Li R.
        An Altered Microbiota in the Lower and Upper Female Reproductive Tract of Women with Recurrent Spontaneous Abortion.
        Microbiol. Spectr. 2022; (Online ahead of print)https://doi.org/10.1128/spectrum.00462-22
        • Liu Y.
        • Ko E.Y.L.
        • Wong K.K.W.
        • Chen X.
        • Cheung W.C.
        • Law T.S.M.
        • Chung J.P.W.
        • Tsui S.K.W.
        • Li T.C.
        • Chim S.S.C.
        Endometrial microbiota in infertile women with and without chronic endometritis as diagnosed using a quantitative and reference range-based method.
        Fertil. Steril. 2019; 112: 707-717
        • Lopes dos Santos Santiago G.
        • Tency I.
        • Verstraelen H.
        • Verhelst R.
        • Trog M.
        • Temmerman M.
        • Vancoillie L.
        • Decat E.
        • Cools P.
        • Vaneechoutte M.
        Longitudinal qPCR Study of the Dynamics ofL. crispatus, L. iners, A. vaginae, (Sialidase Positive)G. vaginalis, andP. bivia in the Vagina.
        PLoS ONE. 2012; 7: e45281
        • Lund M.
        • Kamper-Jørgensen M.
        • Nielsen H.S.
        • Lidegaard Ø.
        • Andersen A.M.N.
        • Christiansen O.B.
        Prognosis for live birth in women with recurrent miscarriage: What is the best measure of success?.
        Obstetrics and Gynecology. 2012; 119https://doi.org/10.1097/AOG.0b013e31823c0413
        • MacIntyre D.A.
        • Chandiramani M.
        • Lee Y.S.
        • Kindinger L.
        • Smith A.
        • Angelopoulos N.
        • Lehne B.
        • Arulkumaran S.
        • Brown R.
        • Teoh T.G.
        • Holmes E.
        • Nicoholson J.K.
        • Marchesi J.R.
        • Bennett P.R.
        The vaginal microbiome during pregnancy and the postpartum period in a European population.
        Sci. Rep. 2015; 5: 8988
        • Martin M.
        Cutadapt removes adapter sequences from high-throughput sequencing reads.
        EMBnet J. 2011; 17https://doi.org/10.14806/ej.17.1.200
        • McQueen D.B.
        • Maniar K.P.
        • Hutchinson A.
        • Confino R.
        • Bernardi L.
        • Pavone M.E.
        Redefining chronic endometritis: the importance of endometrial stromal changes.
        Fertil. Steril. 2021; 116: 855-861
        • McQueen D.B.
        • Perfetto C.O.
        • Hazard F.K.
        • Lathi R.B.
        Pregnancy outcomes in women with chronic endometritis and recurrent pregnancy loss.
        Fertil. Steril. 2015; 104: 927-931
        • Miles S.M.
        • Hardy B.L.
        • Merrell D.S.
        Investigation of the microbiota of the reproductive tract in women undergoing a total hysterectomy and bilateral salpingo-oopherectomy.
        Fertil. Steril. 2017; 107: 813-820
        • Mitchell C.M.
        • Haick A.
        • Nkwopara E.
        • Garcia R.
        • Rendi M.
        • Agnew K.
        • Fredricks D.N.
        • Eschenbach D.
        Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.
        Am. J. Obstet. Gynecol. 2015; 212 (611.e1-611.e9)
        • Moffett A.
        • Shreeve N.
        First do no harm: Uterine natural killer (NK) cells in assisted reproduction.
        Hum. Reprod. 2015; 30: 1519-1525
        • Molina N.M.
        • Sola-Leyva A.
        • Haahr T.
        • Aghajanova L.
        • Laudanski P.
        • Castilla J.A.
        • Altmäe S.
        Analysing endometrial microbiome: Methodological considerations and recommendations for good practice.
        Hum. Reprod. 2021; 36: 859-879
        • Moreno I.
        • Codoñer F.M.
        • Vilella F.
        • Valbuena D.
        • Martinez-Blanch J.F.
        • Jimenez-Almazán J.
        • Alonso R.
        • Alamá P.
        • Remohí J.
        • Pellicer A.
        • Ramon D.
        • Simon C.
        Evidence that the endometrial microbiota has an effect on implantation success or failure.
        Am. J. Obstet. Gynecol. 2016; 215: 684-703
        • Moreno I.
        • Garcia-Grau I.
        • Perez-Villaroya D.
        • Gonzalez-Monfort M.
        • Bahçeci M.
        • Barrionuevo M.J.
        • Taguchi S.
        • Puente E.
        • Dimattina M.
        • Lim M.W.
        • Meneghini G.
        • Aubuchon M.
        • Leondires M.
        • Izquierdo A.
        • Perez-Olgiati M.
        • Chavez A.
        • Seethram K.
        • Bau D.
        • Gomez C.
        • Valbuena D.
        • Vilella F.
        • Simon C.
        Endometrial microbiota composition is associated with reproductive outcome in infertile patients.
        Microbiome. 2022; 10https://doi.org/10.1186/s40168-021-01184-w
        • Nasioudis D.
        • Forney L.J.
        • Schneider G.M.
        • Gliniewicz K.
        • France M.
        • Boester A.
        • Sawai M.
        • Scholl J.
        • Witkin S.S.
        Influence of Pregnancy History on the Vaginal Microbiome of Pregnant Women in their First Trimester.
        Sci. Rep. 2017; 7: 10201
        • Oberle A.
        • Urban L.
        • Falch-Leis S.
        • Ennemoser C.
        • Nagai Y.
        • Ashikawa K.
        • Ulm P.A.
        • Hengstschläger M.
        • Feichtinger M.
        16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.
        Reprod. Biomed. Online. 2021; 42: 1097-1107
        • O'Callaghan J.L.
        • Turner R.
        • Dekker Nitert M.
        • Barrett H.L.
        • Clifton V.
        • Pelzer E.S.
        Re-assessing microbiomes in the low-biomass reproductive niche.
        BJOG. 2020; 127: 147-158
      2. Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., Minchin, P.R., O'Hara, R.B., Simpson, G.L., Solymos, P., Stevens, M.H.H., Szoecs, E., Wagner, H., 2020. Vegan: Community Ecology Package. R package version 2.5-7 [WWW Document].https://CRAN.R-project.org/package=vegan.

        • Petrova M.I.
        • Reid G.
        • Vaneechoutte M.
        • Lebeer S.
        Lactobacillus iners: Friend or Foe?.
        Trends Microbiol. 2017; 25: 182-191
        • Rai R.
        • Regan L.
        Recurrent miscarriage.
        Lancet. 2006; 368: 601-611
        • Ralph S.G.
        • Rutherford A.J.
        • Wilson J.D.
        Influence of bacterial vaginosis on conception and miscarriage in the first trimester: Cohort study.
        BMJ. 1999; 319: 220-223
        • Romero R.
        • Hassan S.S.
        • Gajer P.
        • Tarca A.L.
        • Fadrosh D.W.
        • Nikita L.
        • Galuppi M.
        • Lamont R.F.
        • Chaemsaithong P.
        • Miranda J.
        • Chaiworapongsa T.
        • Ravel J.
        The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women.
        Microbiome. 2014; 2https://doi.org/10.1186/2049-2618-2-4
        • Salter S.J.
        • Cox M.J.
        • Turek E.M.
        • Calus S.T.
        • Cookson W.O.
        • Moffatt M.F.
        • Turner P.
        • Parkhill J.
        • Loman N.J.
        • Walker A.W.
        Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.
        BMC Biology. 2014; 12https://doi.org/10.1186/s12915-014-0087-z
      3. Saqib, S., 2021. Taxonomic annotations – BLAST alignment and text-mining based filtration in R [WWW Document].https://github.com/SchahzadSaqib/taxminer.

        • Shiroda M.
        • Manning S.D.
        Lactobacillus strains vary in their ability to interact with human endometrial stromal cells.
        PLoS One. 2020; 15e0238993
        • Smith S.D.
        • Dunk C.E.
        • Aplin J.D.
        • Harris L.K.
        • Jones R.L.
        Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.
        Am. J. Pathol. 2009; 174: 1959-1971
        • Tu Y.
        • Zheng G.
        • Ding G.
        • Wu Y.
        • Xi J.
        • Ge Y.
        • Gu H.
        • Wang Y.
        • Sheng J.
        • Liu X.
        • Jin L.
        • Huang H.
        Comparative Analysis of Lower Genital Tract Microbiome Between PCOS and Healthy Women.
        Front. Physiol. 2020; 11: 1108
        • Verstraelen H.
        • Vilchez-Vargas R.
        • Desimpel F.
        • Jauregui R.
        • Vankeirsbilck N.
        • Weyers S.
        • Verhelst R.
        • Sutter P.de
        • Pieper D.H.
        • de Wiele T.van
        Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.
        PeerJ. 2016; 4: e1602
        • Virtanen S.
        • Rantsi T.
        • Virtanen A.
        • Kervinen K.
        • Nieminen P.
        • Kalliala I.
        • Salonen A.
        Vaginal Microbiota Composition Correlates Between Pap Smear Microscopy and Next Generation Sequencing and Associates to Socioeconomic Status.
        Sci. Rep. 2019; 9: 7750-7758
      4. Virtanen, S., Saqib, S., Kanerva, T., Nieminen, P., Kalliala, I., Salonen, A., 2021. Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiota and Mycobiota in Healthy Women.https://www.researchsquare.com/article/rs-321778/v1

        • Walther-António M.R.S.
        • Chen J.
        • Multinu F.
        • Hokenstad A.
        • Distad T.J.
        • Cheek E.H.
        • Keeney G.L.
        • Creedon D.J.
        • Nelson H.
        • Mariani A.
        • Chia N.
        Potential contribution of the uterine microbiome in the development of endometrial cancer.
        Genome Med. 2016; 8: 122
        • Wang J.
        • Li Z.
        • Ma X.
        • Du L.
        • Jia Z.
        • Cui X.
        • Yu L.
        • Yang J.
        • Xiao L.
        • Zhang B.
        • Fan H.
        • Zhao F.
        Translocation of vaginal microbiota is involved in impairment and protection of uterine health.
        Nat. Commun. 2021; 12: 4191-4198
        • Wang W.
        • Sung N.
        • Gilman-Sachs A.
        • Kwak-Kim J.
        T Helper (Th) Cell Profiles in Pregnancy and Recurrent Pregnancy Losses: Th1/Th2/Th9/Th17/Th22/Tfh Cells.
        Front Immunol. 2020; 11: 2025
        • 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: 15
        • Winters A.D.
        • Romero R.
        • Gervasi M.T.
        • Gomez-Lopez N.
        • Tran M.R.
        • Garcia-Flores V.
        • Pacora P.
        • Jung E.
        • Hassan S.S.
        • Hsu C.D.
        • Theis K.R.
        Does the endometrial cavity have a molecular microbial signature?.
        Sci. Rep. 2019; 9: 9905

      Biography

      Pirkko Peuranpää is conducting her PhD studies at the University of Helsinki. Her research focuses on understanding the aetiology of recurrent pregnancy loss. She is a general gynaecologist and reproductive medicine specialist who has cared for patients with infertility at Helsinki and Uusimaa District Hospital, Finland.
      Key message
      Lactobacillus crispatus was less abundant, and Gardnerella vaginalis more abundant, in endometrial samples from 47 women with recurrent pregnancy loss compared with 39 healthy control women. Dysbiotic endometrial microbiota may be a novel risk factor for recurrent pregnancy loss, a condition that currently often remains unexplained after standard examinations.