[go: up one dir, main page]

WO2016095789A1 - Détection de taxons bactériens pour la prédiction d'une naissance avant terme après une intervention clinique - Google Patents

Détection de taxons bactériens pour la prédiction d'une naissance avant terme après une intervention clinique Download PDF

Info

Publication number
WO2016095789A1
WO2016095789A1 PCT/CN2015/097341 CN2015097341W WO2016095789A1 WO 2016095789 A1 WO2016095789 A1 WO 2016095789A1 CN 2015097341 W CN2015097341 W CN 2015097341W WO 2016095789 A1 WO2016095789 A1 WO 2016095789A1
Authority
WO
WIPO (PCT)
Prior art keywords
lactobacillus
bacterial
cerclage
acinetobacter
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2015/097341
Other languages
English (en)
Inventor
Stephen Siu-Chung CHIM
Keun-Young Lee
Karen Ka-Wing WONG
Meng MENG
Tak-Yeung LEUNG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chinese University of Hong Kong CUHK
Original Assignee
Chinese University of Hong Kong CUHK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chinese University of Hong Kong CUHK filed Critical Chinese University of Hong Kong CUHK
Publication of WO2016095789A1 publication Critical patent/WO2016095789A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • a prematurely shortened cervix (cervical shortening, CS) and/or a dilated cervix (advanced cervical dilation, ACD) with no regular uterine contraction and no rupture of membrane in the second, instead of the third, trimester are cardinal features in defining cervical insufficiency (CI) in pregnant women.
  • IAI subclinical intraamniotic infection
  • CI patients with subclinical intraamniotic infection subjects the mother and her fetus to morbidity and even mortality risks, which may outweigh the potential benefit of the intervention.
  • a pre-intervention amniocentesis to test for IAI is recommended to select CI patients who may benefit from such intervention.
  • the tests for subclinical IAI rely on amniocentesis for the procurement of amniotic fluid and involve a small but finite risk of procedural-related fetal loss.
  • ACD patients with intra-amniotic infection are associated with poor cerclage outcomes.
  • 17 (51.5% ) were tested positive for bacteria in the amniotic fluid according to Gram stain examination and culture for mycoplasmas (Romero et al. 1992) .
  • only 25% (2/8) of patients who had cerclage with a negative amniotic fluid culture resulted in PTB ⁇ 34 weeks.
  • preterm rupture of membranes occurred in 50% (2/4) of patients who had cerclage and positive amniotic fluid culture, but only in 25% of patients who had cerclage and negative amniotic fluid culture.
  • pre-cerclage amniocentesis might help select patients who will benefit most from cerclage and eliminate from consideration those who will likely not benefit (Berghella et al., 2013, Am J Obstet Gynecol 209 (3) : 181-92) .
  • the current methods are not highly sensitive for testing infection.
  • Mays’s tudy above 2 of 11 (18% ) cases which were tested negative in Gram stain examination, culture and biomarkers of amniotic fluid resulted in PTB ⁇ 28 weeks and their placentas were histologically positive for infection (Mays et al. 2000) .
  • Romero’s study above 2 of 8 (25% ) patients who were tested negative in Gram stain examination and culture of amniotic fluid and had cerclage resulted in PTB ⁇ 34 weeks and preterm rupture of membrane.
  • the current detection methods are only moderately sensitive and target only a limited selection of bacteria. Hence, methods for detecting infection at higher sensitivity and wider coverage of targeted micro-organisms are much awaited in this field, where infection plays important roles in ACD, preterm labor and PTB.
  • the present invention is based, in part, on the discovery of a list of bacterial taxa (genus/species) which are differentially abundant between those CI patients who may benefit and who may not benefit from such intervention to prevent PTB (e.g., surgical cerclage or cervical pessary) .
  • PTB e.g., surgical cerclage or cervical pessary
  • the differential abundance of bacterial taxa in a pregnant patient can be used to predict adverse outcomes due to an intervention, such as the rate of spontaneous PTB ⁇ 34 weeks, the rate of PTB ⁇ 37 weeks and latency (i.e. the days elapsed between intervention and delivery) .
  • the present invention provides methods for determining the risk of an adverse pregnancy outcome for a pregnant subject.
  • the method includes (a) detecting in a biological sample taken from the subject the level of bacteria belonging to at least three bacterial taxon selected from the group consisting of Sneathia sanguinegens, Megasphaera cerevisiae, Gardnerella vaginalis, Prevotella bivia, Prevotella amnii, Parvimonas micra, Mycoplasma hominis, Lactobacillus iners, Ureaplasma parvum, Ureaplasma urealyticum, Aerococcus christensenii, Saccharofermentans acetigenes, Anaerococcus prevotii, Anaerococcus tetradius, Prevotella timonensis, Streptococcus anginosus, Streptococcus constellatus, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius, Parvibacter ca
  • At least one of the at least three bacterial taxa is selected from the group consisting of Sneathia sanguinegens, Megasphaera cerevisiae, Gardnerella vaginalis, Prevotella bivia, Prevotella amnii, Parvimonas micra, Mycoplasma hominis, Lactobacillus iners, Ureaplasma parvum, Ureaplasma urealyticum, Aerococcus christensenii, Saccharofermentans acetigenes, Anaerococcus prevotii, Anaerococcus tetradius, Prevotella timonensis, Streptococcus anginosus, Streptococcus constellatus, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius, Parvibacter caecicola, Atopobium vaginae, Acinetobacter bereziniae, Acinetobacter organizerri, Acinetobacter
  • At least one of the at least three bacterial taxa is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus crispatus , Lactobacillus gallinarum, Corynebacterium tuberculostearicum, Lactobacillus fornicalis, Lactobacillus jensenii, Lactobacillus antri, Lactobacillus frumenti, Lactobacillus oris, Lactobacillus panis, Lactobacillus reuteri, Pseudomonas japonica, Varibaculum cambriense, Alloscardovia omnicolens, Anaerococcus hydrogenalis, and a bacterial taxon specified in Table 2, 3, 4, 5, 7, 8, 9 or 10; and is decreased compared to the standard control level.
  • At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 different bacterial taxa can be detected.
  • an increased risk of adverse pregnancy outcome or adverse pregnancy outcome after surgical cerclage is indicated if 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, or 13 bacterial taxa are increased and 0, 1, 2, 3, 4, 5, or 6 bacterial taxa are decreased.
  • a ratio of bacteria taxa that are increased versus decreased (increased: decreased) indicates that the subject is at risk of having an adverse pregnancy outcome (e.g., spontaneous preterm birth, preterm birth and short latency) after surgical cerclage or pessary ring placement.
  • an adverse pregnancy outcome e.g., spontaneous preterm birth, preterm birth and short latency
  • the subject has an increased risk for adverse pregnancy outcome if the difference between, the ratio of, the sum of, or the product of the total level of bacterial taxa belonging to a first group and total level of bacteria taxa belonging to a second group as set forth in Tables 2-4, is increased or decreased compared to the corresponding value of the standard control.
  • the method further comprises determining a prediction score based on the level of the at least three bacterial taxa.
  • the increase of the level of an individual OTU can be used to predict an adverse pregnancy outcome.
  • an increase of the difference between or ratio of the total level of selected OTUs in a first group (e.g., Group A, such as the total level of 8 increased taxa in Figures 2E and 2F) and the total level of other selected OTUs in a second group e.g., Group B, such as the total level of 4 increased taxa in Figures 2E and 2F
  • a first group e.g., Group A, such as the total level of 8 increased taxa in Figures 2E and 2F
  • Group B such as the total level of 4 increased taxa in Figures 2E and 2F
  • determining the prediction score includes calculating the sum of the levels of the selected taxa after an antilog 10 transformation (i.e., reverting values in the log-scale back to the linear scale) (e.g., Figures 3C and 3D, 3E and 3F) .
  • the subject is a pregnant woman between about 13 weeks to about 37 weeks of gestation.
  • the biological sample is a cervical swab sample, a vaginal swab sample, an amniotic fluid sample, a maternal blood sample (maternal whole blood sample) , a maternal serum sample, a maternal plasma sample, a maternal buccal swab sample or a cervical mucus sample.
  • the method of the present invention can include extracting nucleic acids from the biological sample prior to step (a) .
  • the detecting step comprises detecting the presence of a 16S RNA gene from the bacterial taxon specified in Table 2, 3, 4, 5, 7, 8, 9 or 10.
  • the detecting step can include detecting that the bacterial taxon is taxonomically classified as any species specified in Table 2, 3, 4, 5, 7, 8, 9 or 10.
  • the detecting step can include a polynucleotide amplification assay, hybridization assay or sequencing assay.
  • the amplification assay can be a polymerase chain reaction (PCR) assay. In some instances, the PCR assay is a quantitative PCR assay.
  • the hybridization assay can be an in situ hybridization assay and/or a branched DNA-based detection assay.
  • the sequencing assay can be a sequencing-based assay, primer-extension assay, and/or a mass-spectrometry assay.
  • the adverse pregnancy outcome comprises spontaneous preterm birth (sPTB) at ⁇ 34 weeks, preterm birth (PTB) at ⁇ 37 weeks, or short latency ⁇ 28 days after clinical intervention.
  • the clinical intervention can be the use of a surgical cerclage or the use of a pessary ring around the subject’s cervix.
  • the method also includes determining that the subject has a risk of having advanced cervical dilation or premature cervical shortening if the level of bacteria belonging the at least three bacterial taxon is increased compared to the standard control level.
  • the method also includes determining that the subject will not benefit from clinical intervention, such as cerclage or placement of a pessary ring, to prevent preterm birth if the level of bacteria belonging the at least one bacterial taxon is increased compared to the standard control level.
  • An intervention step other than surgical cerclage therefore can be performed (administered) .
  • the method of the present invention can be used to determine that an intervention step other than surgical cerclage can be performed on a pregnant subject with cervical insufficiency.
  • kits for determining the risk of having an adverse pregnancy outcome in a pregnant subject includes (a) a standard control that provides a biological sample taken from a pregnant subject containing bacteria belonging to at least one bacterial taxon selected from the group consisting of Sneathia sanguinegens, Megasphaera cerevisiae, Gardnerella vaginalis, Prevotella bivia, Prevotella amnii, Parvimonas micra, Mycoplasma hominis, Lactobacillus iners, Ureaplasma parvum, Ureaplasma urealyticum, Aerococcus christensenii, Saccharofermentans acetigenes, Anaerococcus prevotii, Anaerococcus tetradius, Prevotella timonensis, Streptococcus anginosus, Streptococcus constellatus, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius
  • the agent is one or more oligonucleotide primers that specifically hybridizes to and amplify a polynucleotide of the at least one bacterial taxon in an amplification assay.
  • the agent is a polynucleotide probe that specifically hybridizes to a polynucleotide sequence of the at least one bacterial taxon.
  • the kit can include an instruction manual.
  • Figures 1A, 1B, 1C, 1D, 1E and 1F illustrate the use of one or more differentially abundant bacterial taxa or operational taxonomic units (OTUs) to distinguish pregnant patients with a cervical insufficiency (CI) who experienced spontaneous preterm birth at ⁇ 34 weeks gestation after cerclage intervention and those who experienced term birth after cerclage.
  • Figure 1A illustrates the scatter plot of log 10 (relative abundance) or LRA of OTU#1 in CI patients resulting in sPTB ⁇ 34 weeks after cerclage and those resulting in term birth on or >37 weeks (TB) .
  • Middle line and error bars are drawn to the mean and the 95% confidence interval.
  • Figure 1B illustrates the ROC (receiver operating characteristic) curve of LRA of OTU#1 for predicting spontaneous preterm birth (sPTB) after cerclage.
  • Figure 1C shows the scatter plot of LRA (1i OTU) –LRA (5d OTUs) which denotes the LRA of 1increased OTU in the SPTB. NBR. Tev list in Table 2 (i.e., OTU #2) minus the sum of LRA of 5 decreased OTUs (i.e., OTU #159, #104, #74, #44 and #1) .
  • Figure 1D shows the ROC curve of LRA (1i OTU) –LRA (5d OTUs) .
  • Figure 1E shows the scatter plot of LRA (13i OTUs) –LRA (4d OTUs) .
  • Figure 1F shows the ROC curve of LRA (13i OTUs) –LRA (4d OTUs) .
  • Figures 2A, 2B, 2C, 2D, 2E and 2F show that differentially abundant bacterial taxa or operational taxonomic units (OTUs) were identified in CI patients resulting in preterm birth ⁇ 34 weeks (i.e., spontaneous preterm birth and indicated preterm birth due to fetal or maternal complications) after cerclage versus CI patients resulting in term birth on or >37 weeks of gestation after cerclage.
  • Figure 2A illustrates the scatter plot of log 10 (relative abundance) or LRA of OTU#1 in CI patients resulting in sPTB ⁇ 34 weeks after cerclage and those resulting in term birth on or >37 weeks (TB) .
  • LRA of 0, -1, -2, -3, and -4 are equivalent to relative abundance of 100% , 10% , 1% , 0.1% , and 0.01% , respectively.
  • Middle line and error bars are drawn to the mean and the 95% confidence interval.
  • Figure 2B illustrates the ROC (receiver operating characteristic) curve of LRA of OTU#1 for predicting spontaneous preterm birth (sPTB) after cerclage.
  • Figure 2C shows the scatter plot of LRA (1i OTU) –LRA (6d OTUs) which denotes the LRA of 1 increased OTU (i.e., OTU #2) minus the sum of the LRA of 6 decreased OTUs (i.e., OTU #159, #74, #5, #1, #104, and #19) in the “PTB. NBR. Tmisd” list (Table 3) .
  • Figure 2D shows the ROC curve of LRA (1i OTU) –LRA (6d OTUs) .
  • Figure 2E shows the scatter plot of LRA (8i OTUs) –LRA (4d OTUs) .
  • Figure 2F shows the ROC curve of LRA(8i OTUs) –LRA (4d OTUs) .
  • Figures 3A, 3B, 3C, 3D, 3E and 3F show that differentially abundant bacterial taxa or operational taxonomic units (OTUs) were found in CI patients who experienced a short latency interval (i.e., ⁇ 28 days between cerclage intervention and delivery) and those who experienced a long latency interval (i.e., at least 28 days between cerclage intervention and delivery) .
  • Figure 3A illustrates the scatter plot of log 10 (relative abundance) or LRA of OTU#4.
  • Figure 3B illustrates the ROC (receiver operating characteristic) curve of LRA of OTU#4 for predicting spontaneous preterm birth (sPTB) after cerclage.
  • Figure 3C shows the scatter plot of the sum of relative abundance or RA of 4 increased OTU divided by the RA of 1 decreased OTU (RA (4i OTU) /RA (1d OTU) ) . Middle line and error bars are drawn to the mean and the 95% confidence interval.
  • Figure 3D shows the ROC curve of RA (4i OTUs) /RA (1d OTU) .
  • Figure 3E shows the scatter plot of RA (2i OTUs) /RA (2d OTUs) , which denotes the sum of RA of 2 increased OTUs minus the sum of RA of 2 decreased OTUs in the “SLAT. NBR. Tuv” list (Table 4) .
  • Figure 3F shows the ROC curve of RA (2i OTUs) /RA (2d OTUs) .
  • Figures 4A and 4B show that differentially abundant bacterial taxa or operational taxonomic units (OTUs) were found between CI patients who experienced sPTB after cerclage/pessary and those with TB after cerclage/pessary.
  • Figure 4A illustrates the scatter plot of log 10 (abundance) of 9 increased OTUs (LA (9 iOTUs) ) from Table 7. Middle line and error bars are drawn to the mean and the 95% confidence interval. Details of the 9 iOTUs are provided in Tables 7 and 10.
  • Figure 4B illustrates the ROC curve of LRA of (LA (9 iOTUs) for predicting spontaneous preterm birth after clinical intervention (i.e., placement of cerclage or pessary ring) .
  • Figures 5A and 5B show that differentially abundant bacterial taxa or operational taxonomic units (OTUs) were found between CI patients who experienced PTB after cerclage/pessary and those with TB after cerclage/pessary.
  • Figure 5A illustrates the scatter plot of log 10 (relative abundance) of 5 increased OTUs minus LRA of 3 decreased OTUs from Tables 8 and 10.
  • LRA of 0, -1, -2, -3 and -4 are equivalent to relative abundance of 100% , 10% , 1% , 0.1% , and 0.01% , respectively.
  • Middle line and error bars are drawn to the mean and the 95% confidence interval.
  • Figure 5B illustrates the ROC curve of LRA (5iOTUs) –LRA (d3OTUs) in predicting preterm birth after clinical intervention (i.e., placement of cerclage or pessary ring) .
  • Figures 6A and 6B show that differentially abundant bacterial taxa or operational taxonomic units (OTUs) were found between CI patients who experienced short latency after cerclage/pessary and those with long latency after cerclage/pessary.
  • Figure 6A illustrates the scatter plot of log 10 (relative abundance) of 2 increased OTUs minus LRA of 4 decreased OTUs from Tables 9 and 10.
  • Figure 6B illustrates the ROC curve of LRA (2iOTUs) –LRA (4dOTUs) in predicting latency after clinical intervention (i.e., placement of cerclage or pessary ring) .
  • Figures 7A, 7B, 7C, 7D, and 7E show data of the bacterial microbiome of cervical swab samples obtained from 25 cervical insufficiency (CI) patients before cerclage/pessary treatment.
  • Figure 7A shows the clinical outcomes of 25 cervical insufficiency (CI) patients after treatment.
  • Each column represents a patient (P1-P25) in ascending order of gestational age (GA) at delivery.
  • RDS respiratory distress syndrome.
  • BPD bronchopulmonary dysplasia.
  • IVH intraventricular haemorrhage.
  • ROP retinopathy of prematurity.
  • Neonatal death death within 28 days after delivery.
  • Y Yes; n, no; -, not determined.
  • the 10 most abundant bacterial taxa in the “sPTB after treatment” CI cervices are shown in Figure 7B.
  • the 10 most abundant bacterial taxa in the “TB after treatment” CI cervices are shown in Figure 7C. Shown values are the log of 10 the abunance values (normalized sequencing read counts, the Cumulative Sum Scaling (CSS) method) of each bacterial taxon (row) in the cervical swab sample of each patient (column) .
  • CCS Cumulative Sum Scaling
  • Each row represents an operational taxonomic unit (Otu) formed by clustering sequences of ⁇ 97% identity.
  • Otu is taxonomically classified at the genus level using the Ribosomal Database Project (RDP) Bayesian rRNA Classifier (Version 2.9, September 2014, RDP 16S rRNA training set 10) .
  • Lactobacilli are further matched against the 16S rRNA database (GenBank) using BLAST (highest score) and MOLEBLAST (best multiple-alignment of BLAST matches) for deriving the species information.
  • FDR False Discovery Rate
  • Figures 8A, 8B, 8C, and 8D show data of the 7 selected bacteria taxa of the LA7 values in cervical insufficiency (CI) patients.
  • Figure 8A shows LA7 values (the total abundance of the selected bacterial taxa in logarithmic (base 10) scale) in two groups of cervical insufficiency (CI) patients both receiving treatment but with different outcomes.
  • Cervical swab samples for measuring the LA7 were collected from CI patients before the cerclage/pessary treatment. After the treatment, 10 patients resulted in spontaneous preterm birth ⁇ 34 weeks (the “sPTB after treatment” group, circles) , and 15 patients resulted in term birth ⁇ 37 weeks (the “TB after treatment” group, triangles) .
  • Figure 7 shows a receiver operating characteristic curve of LA7 in distinguishing CI patients results in the “sPTB after treatment” form those resultsing in “TB after treatment. ”
  • Figure 8C shows Kaplan-Meier curves of the proportion of continued pregnancies at different gestational period in CI patients with LA7 ⁇ 2.26 (negative) and those with LA7 ⁇ 2.26 (positive) .
  • Figure 8D shows Kaplan-Meier curves of the proportion of continued pregnancies at different days after treatment in LA7-positive and LA-negative CI patients.
  • Log-rank (Mantel-Cox) test has shown that the LA7-positive patients were more likely to delivered in a shorter time interval after treatment (latency period, i.e., days between treatment and delivery) , compared to LA7-negative patients, with the median latency period of 17 days vs. 129 days (p ⁇ 0.0001, Hazard Ratio (logrank) 5.74, 95% confidence interval, 15.5 to 202) .
  • non-invasive methods and kits for determining whether a pregnant subject with cervical insufficiency is likely to have an adverse pregnancy outcome such as spontaneous PTB ⁇ 34 weeks, preterm birth ⁇ 37 weeks and short latency, after clinical intervention to prevent preterm birth.
  • the method includes measuring the level (e.g., amount or abundance) of one or more bacterial taxa in a sample from the subject, and determining if the level of the one or more bacterial taxa is increased or decreased compared to a standard control level.
  • an increased level of particular bacterial taxa indicates that the subject is likely to experience an adverse pregnancy outcome.
  • a decreased level of particular bacterial taxa indicates that the subject is likely to experience an adverse pregnancy outcome.
  • an adverse pregnancy outcome refers to a condition that reduces the chance of delivering/birthing a healthy baby.
  • Non-limiting examples of an adverse pregnancy outcome includes multiple first trimester miscarriages, a second trimester pregnancy loss, preterm birth (e.g., spontaneous or indicated) , preterm pre-clampsia, preterm clampsia, fetal growth restriction, abruption placenta, fetal death/stillbirth, birth defects, Apgar score at 1 minute of ⁇ 7, Apgar score at 5 minute of ⁇ 7, clinical chorioamnioitis, pathological chorioamnioitis, neonatal respiratory distress syndrome, neonatal bronchopulmonary dysplasia, neonatal sepsis, neonatal intraventricular hemorrhage, etc.
  • bacterial taxon refers to the taxonomy, i.e., the rank-based classification of bacteria.
  • the hierarchical biological classification includes life, domain, kingdom, phylum, class, order, family, genus and species.
  • biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes, or processed forms of any of such samples.
  • Biological samples include a cervical swab, a vaginal swab, a uterine swab, blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like) , sputum or saliva, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, a biopsy tissue etc.
  • a biological sample is typically obtained from a eukaryotic organism, which may be a mammal, may be a primate and may be a human subject.
  • biopsy refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., cervix, vagina, tongue, colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, stomach tissue, etc. ) among other factors. Representative biopsy techniques include, but are not limited to, a swab biopsy, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy and may comprise colonoscopy. A wide range of biopsy techniques are well known to those skilled in the art who will choose between them and implement them with minimal experimentation.
  • isolated nucleic acid molecule means a nucleic acid molecule that is separated from other nucleic acid molecules that are usually associated with the isolated nucleic acid molecule.
  • an "isolated" nucleic acid molecule includes, without limitation, a nucleic acid molecule that is free of nucleotide sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion) .
  • an isolated nucleic acid molecule can be introduced into a vector (e.g., a cloning vector or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule.
  • an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, single nucleotide polymorphisms (SNPs) , and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem.
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens) , wherein the amino acid residues are linked by covalent peptide bonds.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may include those having non-naturally occurring D-chirality, as disclosed in WO01/12654, which may improve the stability (e.g., half-life) , bioavailability, and other characteristics of a polypeptide comprising one or more of such D-amino acids. In some cases, one or more, and potentially all of the amino acids of a therapeutic polypeptide have D-chirality.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • the terms “identical” or percent “identity, ” in the context of describing two or more polynucleotide or amino acid sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (for example, a variant of a bacterial protein or interest used in the method of this invention (e.g., for predicting adverse pregnancy outcomes) has at least 80% sequence identity, preferably 85% , 90% , 91% , 92% , 93, 94% , 95% , 96% , 97% , 98% , 99% , or 100% identity, to a reference sequence, e.g., a corresponding wild-type bacterial protein of interest) , when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • a reference sequence e.g., a corresponding wild-type bacterial protein of interest
  • sequences are then said to be “substantially identical. ”
  • this definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.
  • a “comparison window” includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith &Waterman, Adv. Appl. Math. 2: 482 (1981) , by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol.
  • the same selected contiguous position on the 16S rRNA gene of a bacterial taxon may be reported to have slightly different sequence identity (e.g., 1% or 5% difference) to the same reference segment.
  • sequence identity e.g., 1% or 5% difference
  • Such non-critical discrepancy in the reported sequence identity may occur due to different handling of heading or trailing space or gaps or comparison windows in the alignment used by the algorithms.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra) .
  • These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0) .
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989) ) .
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat’ l. Acad. Sci. USA, 90: 5873-5787 (1993) ) .
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ) , which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P (N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • stringent hybridization conditions and “high stringency” refer to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium) .
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5 x SSC, and 1% SDS, incubating at 42°C, or, 5 x SSC, 1% SDS, incubating at 65°C, with wash in 0.2 x SSC, and 0.1% SDS at 65°C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 1x SSC at 45°C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous references, e.g., Current Protocols in Molecular Biology, ed. Ausubel, et al.
  • an “increase” or a “decrease” refers to a detectable positive or negative change in quantity from a comparison control, e.g., an established standard control (such as an average expression level of a bacterial mRNA or protein found in normal cervical or vaginal tissue from a pregnant control subject) .
  • An increase is a positive change that is typically at least 10% , or at least 20% , or 50% , or 100% , and can be as high as at least 2-fold or at least 5-fold or even 10-fold of the control value.
  • a decrease is a negative change that is typically at least 10% , or at least 20% , 30% , or 50% , or even as high as at least 80% or 90% of the control value.
  • a "polynucleotide hybridization method" as used herein refers to a method for detecting the presence and/or quantity of a pre-determined polynucleotide sequence based on its ability to form Watson-Crick base-pairing, under appropriate hybridization conditions, with a polynucleotide probe of a known sequence. Examples of such hybridization methods include Southern blot, Northern blot, and in situ hybridization.
  • Primers refer to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction (PCR) , to amplify a nucleotide sequence based on the polynucleotide sequence corresponding to a gene of interest, e.g., the cDNA or genomic sequence for a specific bacterial gene or a portion thereof.
  • PCR polymerase chain reaction
  • at least one of the PCR primers for amplification of a polynucleotide sequence is sequence-specific for that polynucleotide sequence. The exact length of the primer will depend upon many factors, including temperature, source of the primer, and the method used.
  • the oligonucleotide primer typically contains at least 10, or 15, or 20, or 25 or more nucleotides, although it may contain fewer nucleotides or more nucleotides.
  • the factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.
  • primer pair means a pair of primers that hybridize to opposite strands a target DNA molecule or to regions of the target DNA which flank a nucleotide sequence to be amplified.
  • primer site means the area of the target DNA or other nucleic acid to which a primer hybridizes.
  • label, ” “detectable label, ” or “ ” detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA) , biotin, digoxigenin, or haptens and proteins that can be made detectable, e.g., by incorporating a radioactive component into the peptide or used to detect antibodies specifically reactive with the peptide.
  • a detectable label is attached to a probe or a molecule with defined binding characteristics (e.g., a polypeptide with a known binding specificity or a polynucleotide) , so as to allow the presence of the probe (and therefore its binding target) to be readily detectable.
  • defined binding characteristics e.g., a polypeptide with a known binding specificity or a polynucleotide
  • Standard control refers to a predetermined amount or concentration of bacteria belonging to a specific bacterial genus, a bacterial polynucleotide or a bacterial polypeptide that is present in an established normal tissue sample, e.g., a normal cervical tissue sample.
  • the standard control value is suitable for the use of a method of the present invention, to serve as a basis for comparing the amount of a specific bacterial genus, mRNA or protein that is present in a test sample.
  • An established sample serving as a standard control provides an average amount of the bacterial genus, mRNA or protein that is typical for a cervical tissue sample of an average, healthy pregnant human with, for example, a closed cervix or normal-length cervix, as conventionally defined.
  • a standard control value may vary depending on the nature of the sample, the manner of sample collection, as well as other factors such as the gender, age, ethnicity of the subjects (and in the case of pregnant women, gestational age) based on whom such a control value is established.
  • the selected group of pregnant humans generally have a similar gestational-age to that of a subject whose cervical tissue sample is tested for indication of a risk of having an adverse pregnancy or neonatal outcome.
  • other factors such as age, ethnicity, medical history are also considered and preferably closely matching between the profiles of the test subject and the selected group of individuals establishing the “average” value.
  • amount refers to the quantity of a bacterial taxon of interest, a bacterial polynucleotide of interest or a bacterial polypeptide of interest present in a sample. Such quantity may be expressed in the absolute terms, i.e., the total quantity of the bacterial taxon, polynucleotide or polypeptide in the sample, or in the relative terms, i.e., the concentration of the bacterial taxon, polynucleotide or polypeptide in the sample.
  • subject includes individuals who seek medical attention due to a potential risk of having an adverse pregnancy outcome or neonatal outcome, e.g., any pregnant individual. Subjects also include individuals who have had an adverse pregnancy or neonatal outcome during a prior pregnancy.
  • abnormal pregnancy outcome refers to a condition in which a pregnant mother experiences preterm labor (e.g., labor ⁇ 37 weeks gestation) or preterm birth (e.g., birth ⁇ 37 weeks gestation) .
  • preterm labor e.g., labor ⁇ 37 weeks gestation
  • preterm birth e.g., birth ⁇ 37 weeks gestation
  • the invention is based, in part, on the discovery of differentially abundant bacterial taxa in the cervical swab samples of women with advanced cervical dilation/cervical shortening and resulting in preterm birth after clinical intercention, compared with those in appropriately-controlled samples from appropriately-matched women without the corresponding condition, i.e. women with advanced cervical dilation/cervical shortening and resulting in term birth after clinical intervention.
  • the increased level of bacteria from particular bacterial taxa e.g., Sneathia sanguinegens, Megasphaera cerevisiae, Gardnerella vaginalis, Prevotella bivia, Prevotella amnii, Parvimonas micra, Mycoplasma hominis, Lactobacillus iners, Ureaplasma parvum, Ureaplasma urealyticum, Aerococcus christensenii, Saccharofermentans acetigenes, Anaerococcus prevotii, Anaerococcus tetradius, Prevotella timonensis, Streptococcus anginosus, Streptococcus constellatus, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius, Parvibacter caecicola, Atopobium vaginae, Acinetobacter bereziniae, Acinetobacter organizerri, Acinetobacter guillouiae,
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp) . These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • bp base pairs
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981) , using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12: 6159-6168 (1984) . Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983) .
  • HPLC high performance liquid chromatography
  • the level of bacteria belonging to a specific bacterial taxon e.g., a bacterial species or genera
  • a specific bacterial taxon e.g., a bacterial species or genera
  • the level of bacteria belonging to a specific bacterial taxon is increased or decreased in correlation with the likelihood of an adverse pregnancy outcome after clinical intervention, such as cervical cerclage or application of a pessary ring.
  • the bacteria taxa include Sneathia sanguinegens, Megasphaera cerevisiae, Gardnerella vaginalis, Prevotella bivia, Prevotella amnii, Parvimonas micra, Mycoplasma hominis, Lactobacillus iners, Ureaplasma parvum, Ureaplasma urealyticum, Aerococcus christensenii, Saccharofermentans acetigenes, Anaerococcus prevotii, Anaerococcus tetradius, Prevotella timonensis, Streptococcus anginosus, Streptococcus constellatus, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius, Parvibacter caecicola, Atopobium vaginae, Acinetobacter bereziniae, Acinetobacter organizerri, Acinetobacter guillouiae, Acinetobacter gyllenbergii,
  • Sneathia sanguinegens can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AJ344093.1 or NR_118342.1.
  • bacteria of the taxon Sneathia sanguinegens are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AJ344093.1 or as having 16S rRNA nucleotide sequence with at least 96% or 97% sequence identity to the sequence of GenBank Accession No. NR_118342.1 (complete sequence) .
  • Megasphaera cerevisiae can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_113307.1.
  • bacteria of the taxon Megasphaera cerevisiae are detected as having 16S rRNA nucleotide sequence with at least 92% or 93% sequence identity to the sequence of GenBank Accession No. NR_113307.1.
  • Gardnerella vaginalis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. EF194095.1.
  • bacteria of the taxon Gardnerella vaginalis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. EF194095.1.
  • Prevotella bivia can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. L16475.1.
  • bacteria of the taxon Prevotella bivia are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. L16475.1.
  • Prevotella amnii can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_113093.1.
  • bacteria of the taxon Prevotella amnii are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_113093.1.
  • Parvimonas micra can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_114338.1.
  • bacteria of the taxon Parvimonas micra are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_114338.1.
  • Mycoplasma hominis bacteria can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_113679.1.
  • bacteria of the taxon Mycoplasma hominis are detected as having a 16S rRNA genomic sequence with 100% sequence identity to the nucleotide sequence of GenBank Accession No. NR_113679.1.
  • Lactobacillus iners can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_036982.1 or NR_102836.1.
  • bacteria of the taxon Lactobacillus iners are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_036982.1 or as having 16S rRNA nucleotide sequence with 100% sequence identity to the sequence of GenBank Accession No. NR_102836.1.
  • Ureaplasma parvum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AB069823.1.
  • bacteria of the taxon Ureaplasma parvum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AB069823.1.
  • Ureaplasma urealyticum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. L08642.1.
  • bacteria of the taxon Ureaplasma urealyticum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. L08642.1.
  • Aerococcus christensenii can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. Y17005.1.
  • bacteria of the taxon Aerococcus christensenii are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. Y17005.1.
  • Saccharofermentans acetigenes can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_115340.1.
  • bacteria of the taxon Saccharofermentans acetigenes are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_115340.1.
  • Anaerococcus prevotii can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AB971807.1.
  • bacteria of the taxon Anaerococcus prevotii are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AB971807.1.
  • Anaerococcus tetradius can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_041941.1.
  • bacteria of the taxon Anaerococcus tetradius are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_041941.1.
  • Prevotella timonensis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. KC311742.1.
  • bacteria of the taxon Prevotella timonensis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. KC311742.1.
  • Streptococcus anginosus can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_118289.1.
  • bacteria of the taxon Streptococcus anginosus are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_118289.1.
  • Streptococcus constellatus can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. JN787163.1.
  • bacteria of the taxon Streptococcus constellatus are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. JN787163.1.
  • Peptoniphilus lacrimalis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AB971812.1 or NR_041938.1.
  • bacteria of the taxon Peptoniphilus lacrimalis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AB971812.1 or as having 16S rRNA nucleotide sequence with 100% sequence identity to the sequence of GenBank Accession No. NR_041938.1.
  • Peptostreptococcus anaerobius can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_118652.1.
  • bacteria of the taxon Peptostreptococcus anaerobius are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_118652.1.
  • Parvibacter caecicola can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_117374.1.
  • bacteria of the taxon Parvibacter caecicola are detected as having 16S rRNA nucleotide sequence with at least 91% or 92% sequence identity to the sequence of GenBank Accession No. NR_117374.1.
  • Atopobium vaginae can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AJ585206.2 or NR_117757.1.
  • bacteria of the taxon Atopobium vaginae are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AJ585206.2 or as having 16S rRNA nucleotide sequence with at least 97% or 98% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_117757.1.
  • Acinetobacter bereziniae can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_117625.1.
  • bacteria of the taxon Acinetobacter bereziniae are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_117625.1.
  • Acinetobacter organizerri can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_117627.1
  • bacteria of the taxon Acinetobacter organizerri are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_117627.1.
  • Acinetobacter guillouiae can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. N NR_117626.1.
  • bacteria of the taxon Acinetobacter guillouiae are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_117626.1.
  • Acinetobacter gyllenbergii can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_042026.1.
  • bacteria of the taxon Acinetobacter gyllenbergii are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_042026.1.
  • Acinetobacter junii can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AB777646.1.
  • bacteria of the taxon Acinetobacter junii are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AB777646.1.
  • Corynebacterium pyruviciproducens can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. GU797881.1.
  • bacteria of the taxon Corynebacterium pyruviciproducens are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. GU797881.1.
  • Tissierella praeacuta can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_119111.1.
  • bacteria of the taxon Tissierella praeacuta are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_119111.1.
  • Gardnerella vaginalis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. EF194095.1
  • bacteria of the taxon Gardnerella vaginalis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. EF194095.1.
  • Bifidobacterium breve can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. M58731.1.
  • bacteria of the taxon Bifidobacterium breve are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. M58731.1.
  • Bifidobacterium choerinum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_037116.1.
  • bacteria of the taxon Bifidobacterium choerinum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_037116.1.
  • Bifidobacterium longum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. U10152.1.
  • bacteria of the taxon Bifidobacterium longum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. U10152.1.
  • Bifidobacterium pseudolongum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. M58742.1.
  • bacteria of the taxon Bifidobacterium pseudolongum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. M58742.1.
  • Lactobacillus acidophilus can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. M99704.1.
  • bacteria of the taxon Lactobacillus acidophilus are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. M99704.1.
  • Lactobacillus crispatus can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AY339181.1.
  • bacteria of the taxon Lactobacillus crispatus are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AY339181.1.
  • Lactobacillus gallinarum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_113261.1.
  • bacteria of the taxon Lactobacillus gallinarum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_113261.1.
  • Corynebacterium tuberculostearicum can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_119173.1.
  • bacteria of the taxon Corynebacterium tuberculostearicum are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_119173.1.
  • Lactobacillus fornicalis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. Y18654.1.
  • bacteria of the taxon Lactobacillus fornicalis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. Y18654.1.
  • Lactobacillus antri can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. AY253659.1.
  • bacteria of the taxon Lactobacillus antri are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. AY253659.1.
  • Lactobacillus frumenti can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_025371.1.
  • bacteria of the taxon Lactobacillus frumenti are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_025371.1.
  • Lactobacillus oris can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_118973.1.
  • bacteria of the taxon Lactobacillus oris are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_118973.1.
  • Lactobacillus panis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. X94230.1.
  • bacteria of the taxon Lactobacillus panis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. X94230.1.
  • Lactobacillus reuteri can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. L23507.1.
  • bacteria of the taxon Lactobacillus reuteri are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. L23507.1.
  • Pseudomonas japonica can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_040992.1.
  • bacteria of the taxon Pseudomonas japonica are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_040992.1.
  • Varibaculum cambriense can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_114873.1.
  • bacteria of the taxon Varibaculum cambriense are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_114873.1.
  • Alloscardovia omnicolens can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_042583.1.
  • bacteria of the taxon Alloscardovia omnicolens are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_042583.1.
  • Anaerococcus hydrogenalis can be identified as having a 16S rRNA nucleotide sequence with at least 90% , e.g., at least 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% or 100% sequence identity to the 16S rRNA nucleotide sequence of GenBank Accession No. NR_113029.1.
  • bacteria of the taxon Anaerococcus hydrogenalis are detected as having 16S rRNA nucleotide sequence with at least 93% or 94% sequence identity to the sequence of GenBank Accession No. NR_113029.1.
  • the method includes detecting the level of at least one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or more bacterial taxa or OTUs in a sample from a pregnant subject.
  • the method includes measuring the level of 2 to 20, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, bacterial taxa in the sample.
  • the method includes measuring one or more OTUs as set forth in Tables 2, 3 or 4.
  • the method can include measuring at least 2 OTUs, e. g, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, as set forth in Tables 2, 3 or 4.
  • the present invention relates to measuring the amount of bacteria of a specific bacteria taxon found in a pregnant woman’s cervix or vagina, especially in a cervical swab or vaginal swab sample, as a means to assess the risk of having an adverse pregnancy outcome or neonatal outcome, such as preterm labor and preterm delivery.
  • the first steps of practicing this invention are to obtain a cervical or vaginal tissue sample from a test subject, such that the nucleic acids, e.g., RNA or DNA, contained in the sample may be analyzed.
  • a biological sample such as cervical or vaginal tissue, cervical mucus, amniotic fluid or maternal blood is obtained from a person to be tested or monitored using a method of the present invention. Collection of cervical or vaginal epithelial cells, cervical mucus, amniotic fluid or maternal blood from an individual is performed in accordance with the standard protocol hospitals or clinics generally follow, such as during a cervical screening. An appropriate amount of cervical or vaginal epithelium, scraped cells, mucus, and/or biological fluid is collected and may be stored according to standard procedures prior to further preparation.
  • the analysis of the bacteria found in a pregnant patient's sample according to the present invention may be performed using, e.g., cells, tissue, mucosa, or fluids found in the sample.
  • the methods for preparing cell, tissue or fluid samples for nucleic acid extraction are well known among those of skill in the art.
  • a subject's cervical or vaginal mucosa sample can be treated to such that bacterial DNA or RNA in the sample can be analyzed.
  • RNA contamination should be eliminated to avoid interference with DNA analysis.
  • Pretreatment of the biological sample with lysis buffer and enzymes, including mutanolysin and proteinase K, can also be used before the extraction.
  • Methods for detecting target DNA include either PCR analysis, quantitative analysis with fluorescence labelling or Southern blot analysis.
  • the target DNA can be the gene encoding the 16S ribosomal RNA (the 16S rRNA gene) , or other genes or genomic sequences of interest possessed by a specific bacterial taxon.
  • PCR polymerase chain reaction
  • PCR amplification is typically used in practicing the present invention, one of skill in the art will recognize that amplification of the relevant genomic sequence may be accomplished by any known method, such as the ligase chain reaction (LCR) , transcription-mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification (NASBA) , each of which provides sufficient amplification. More recently developed branched-DNA technology may also be used to quantitatively determining the amount of specific bacterial mRNA markers. For a detailed description of branched-DNA signal amplification for direct quantitation of nucleic acid sequences in clinical samples, see, for example, Nolte, Adv. Clin. Chem. 33: 201-235, 1998.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • Additional means suitable for detecting a polynucleotide sequence for practicing the methods of the present invention include but are not limited to mass spectrometry, primer extension, polynucleotide hybridization, real-time PCR, melting curve analysis, high resolution melting analysis, heteroduplex analysis, pyrosequencing, and electrophoresis.
  • RNA preparation e.g., described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001
  • various commercially available reagents or kits such as Trizol reagent (Invitrogen, Carlsbad, CA) , Oligotex Direct mRNA Kits (Qiagen, Valencia, CA) , RNeasy Mini Kits (Qiagen, Hilden, Germany) , and Series 9600 TM (Promega, Madison, WI) , may also be used to obtain mRNA from a biological sample from a test subject. Combinations of more than one of these methods may also be used.
  • RNA transcripts of interest that is expressed by bacteria of a specific bacterial taxon may be quantified.
  • the amount of 16S ribosomal RNA (rRNA) for a particular bacterial taxon such as, but not limited to, Sneathia sanguinegens, Megasphaera cerevisiae, Gardnerella vaginalis, Prevotella bivia, Prevotella amnii, Parvimonas micra, Mycoplasma hominis, Lactobacillus iners, Ureaplasma parvum, Ureaplasma urealyticum, Aerococcus christensenii, Saccharofermentans acetigenes, Anaerococcus prevotii, Anaerococcus tetradius, Prevotella timonensis, Streptococcus anginosus, Streptococcus constellatus, Peptoniphilus lacrimalis, Peptostreptococc
  • a DNA copy (cDNA) of a bacterial RNA transcript of interest Prior to the amplification step, a DNA copy (cDNA) of a bacterial RNA transcript of interest must be synthesized. This is achieved by reverse transcription, which can be carried out as a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR) , a modification of the polymerase chain reaction for amplifying RNA.
  • RT-PCR homogeneous reverse transcription-polymerase chain reaction
  • PCR PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems.
  • PCR is most usually carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available.
  • PCR amplification of the target RNA is typically used in practicing the present invention.
  • amplification of these bacterial RNA species in the sample may be accomplished by any known method, such as ligase chain reaction (LCR) , transcription-mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification (NASBA) , each of which provides sufficient amplification.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • More recently developed branched-DNA technology may also be used to quantitatively determining the amount of specific bacterial RNA markers.
  • the bacterial DNA or RNA transcripts of interest can also be detected using other standard techniques, well-known to those of skill in the art. Although the detection step is typically preceded by an amplification step, amplification is not required in the methods of the invention. For instance, the DNA or RNA may be identified by size fractionation (e.g., gel electrophoresis) , whether or not proceeded by an amplification step.
  • size fractionation e.g., gel electrophoresis
  • the presence of a band of the same size as the standard comparison is an indication of the presence of a target DNA or RNA, the amount of which may then be compared to the control based on the intensity of the band.
  • oligonucleotide probes specific to the DNA or RNA of interest can be used to detect the presence of such DNA or RNA species and indicate the amount of DNA or RNA in comparison to the standard comparison, based on the intensity of signal imparted by the probe.
  • Sequence-specific probe hybridization is a well-known method of detecting a particular nucleic acid comprising other species of nucleic acids. Under sufficiently stringent hybridization conditions, the probes hybridize specifically only to substantially complementary sequences. The stringency of the hybridization conditions can be relaxed to tolerate varying amounts of sequence mismatch.
  • hybridization formats well known in the art, including but not limited to, solution phase, solid phase, or mixed phase hybridization assays.
  • the following articles provide an overview of the various hybridization assay formats: Singer et al., Biotechniques, 4: 230, 1986; Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situ Hybridization, Wilkinson ed., IRL Press, Oxford University Press, Oxford; andHames and Higgins eds., Nucleic Acid Hybridization: A Practical Approach, IRL Press, 1987.
  • the hybridization complexes are detected according to well-known techniques.
  • Nucleic acid probes capable of specifically hybridizing to a target nucleic acid i.e., the RNA or the amplified DNA
  • One common method of detection is the use of autoradiography using probes labeled with 3 H, 125 I, 35 S, 14 C, or 32 P, or the like.
  • the choice of radioactive isotope depends on research preferences due to ease of synthesis, stability, and half-lives of the selected isotopes.
  • labels include compounds (e.g., biotin and digoxigenin) , which bind to anti-ligands or antibodies labeled with fluorophores, chemiluminescent agents, and enzymes.
  • probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents or enzymes. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • probes and primers necessary for practicing the present invention can be synthesized and labeled using well known techniques.
  • Oligonucleotides used as probes and primers may be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts., 22: 1859-1862, 1981, using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res. 12: 6159-6168, 1984. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier, J. Chrom., 255: 137-149, 1983.
  • PCR polymerase chain reaction
  • Additional means suitable for detecting a polynucleotide sequence for practicing the methods of the present invention include but are not limited to mass spectrometry, primer extension, polynucleotide hybridization, real-time PCR, melting curve analysis, high resolution melting analysis, heteroduplex analysis, pyrosequencing, and electrophoresis.
  • pregnant women without the adverse pregnancy outcome e.g., preterm birth or spontaneous preterm birth
  • a group of healthy pregnant women, pregnant women who are not at risk of having an adverse pregnancy outcome or neonatal outcome, or pregnant women who are later confirmed to deliver within the normal time frame of their pregnancy, as conventionally defined can also be first selected.
  • the group may include a group of pregnant women who have had a full-term labor and delivery.
  • the individuals are within the appropriate parameters, if applicable, for the purpose of screening for and/or monitoring risk of adverse pregnancy outcomes using the methods of the present invention.
  • the individuals may be of a similar gestational age and comparable health status.
  • the individuals are of similar age, similar ethnic background, similar cervical length, similar cervical dilation status, or are receiving similar clinical intervention.
  • the normal delivery time of the selected individuals will be confirmed later on, and anyone among the selected individuals who turn out to give birth sooner or later than the normal delivery time frame will be excluded from the group to provide data as a “standard control. ”
  • the healthy status of the selected individuals is confirmed by well established, routinely employed methods including but not limited to general physical examination of the individuals and general review of their medical history.
  • the selected group of healthy individuals must be of a reasonable size, such that the average amount/concentration of bacteria of one or more bacterial taxa in the cervical tissue sample obtained from the group can be reasonably regarded as representative of the normal or average level among the general population of healthy pregnant women.
  • the selected group comprises at least 10 pregnant human subjects.
  • an average value for the bacteria of one or more taxa is established based on the individual values found in each subject of the selected healthy control group, this average or median or representative value or profile is considered a standard control. A standard deviation is also determined during the same process. In some cases, separate standard controls may be established for separately defined groups having distinct characteristics such as age, gestational age, or ethnic background.
  • the invention provides compositions and kits for practicing the methods described herein to assess the level of bacteria from one or more specific taxa in a pregnant subject, which can be used for various purposes such as determining the risk of having an adverse pregnancy or neonatal outcome.
  • Kits for carrying out assays for determining the RNA level of bacteria of a bacterial taxon of interest typically include at least one oligonucleotide useful for specific hybridization with at least one segment of a coding sequence of interest or its complementary sequence.
  • this oligonucleotide is labeled with a detectable moiety.
  • the kits may include at least two oligonucleotide primers that can be used in the amplification of at least one segment of a bacterial DNA or RNA transcript of interest by PCR, particularly by RT-PCR.
  • Kits for carrying out assays for determining the protein level of bacteria of a bacterial taxon of interest typically include at least one antibody useful for specific binding to the target protein amino acid sequence.
  • this antibody is labeled with a detectable moiety.
  • the antibody can be either a monoclonal antibody or a polyclonal antibody.
  • the kits may include at least two different antibodies, one for specific binding to the target protein (i.e., the primary antibody) and the other for detection of the primary antibody (i.e., the secondary antibody) , which is often attached to a detectable moiety.
  • kits also include an appropriate standard control.
  • the standard controls indicate the average value of a target protein or a target mRNA expressed by bacteria from a specific bacterial taxon in the cervical epithelium of healthy, pregnant subjects who are not at risk of having an adverse pregnancy or neonatal outcome.
  • standard control may be provided in the form of a set value.
  • the kits of this invention may provide instruction manuals to guide users in analyzing test samples and assessing the risk of having an adverse pregnancy event, such as preterm delivery, in a test subject.
  • Example 1 Methods for predicting pregnancy outcomes in pregnant subjects experiencing cervical insufficiency.
  • PTB Preterm birth
  • CI cervical insufficiency
  • CS prematurely shortened cervix
  • ACD advanced cervical dilation
  • the shortened/dilated cervix may expose the chorioamnionic membranes to bacteria in the lower genital tract, lead to ascending infection into the amniotic cavity (intra-amniotic infection, IAI) , and trigger preterm labor and PTB.
  • IAI intra-amniotic infection
  • amniocentesis itself is invasive and may trigger infection. Since ascending infection is the major route of IAI, we reasoned that the concerned bacteria may be detected at a stage, earlier than IAI, via cervical swab sampling, which is relatively non-invasive.
  • the invention is based, in part, on the systematic measurement of the relative abundance of essentially all kinds of bacteria colonizing the cervices of CI patients, using 16S ribosomal RNA-based massively parallel sequencing.
  • bacterial taxa (genera/species) that are differentially abundant in the dilated cervices between (i) ACD patients undergoing cerclage and resulting in preterm birth ⁇ 34 weeks (PTB after cerclage) , and (ii) ACD patients undergoing cerclage and resulting in term birth (TB after cerclage) (Study A) .
  • PTB after cerclage preterm birth ⁇ 34 weeks
  • TB after cerclage TB after cerclage
  • the detection of the differentially abundant bacteria identified in the study can be used to predict the rate of sPTB ⁇ 34 weeks after intervention based on various tests involving selected members from the list (Table 5) identified in the study above (Study A) .
  • the data from the study can also be used to predict the rate of PTB ⁇ 34 weeks after intervention based on various tests involving selected members from the list identified.
  • the results of the study can also be used to predict the latency, i.e. days elapsed after intervention and delivery, based on various tests involving selected members from the list identified.
  • the methods described herein are useful for predicting the outcomes of intervention, including the rate of sPTB ⁇ 34 weeks, the rate of PTB ⁇ 34 weeks and latency, for the CI patients when selected members of bacterial taxa from the lists identified in the studies described below are present or absent or over-represented or under-represented in relative abundance or absolute quantity (abundance) .
  • the method includes measuring the relative abundance or abundance of the identified taxa in a given sample using MPS or any sequencing-based approach.
  • the method includes measuring the relative abundance or abundance of the identified taxa in a given sample using detection methods involving amplification or nucleotide hybridization, such as quantitative polymerase chain reaction (qPCR) assays or in situ hybridization which specifically targets those taxa.
  • qPCR quantitative polymerase chain reaction
  • pregnancies involving preeclampsia multiple pregnancies, fetal distress, growth restriction, chromosomal or structural abnormalities.
  • we also excluded participants who had sexual activities or applied any used any other vaginal applications e.g., vaginal medication or suppositories, douche) 48 hours before sample collection or on antibiotic or antimycotic drugs 30 days before sample collection, or ovarian tumor.
  • each cervical swab sample was collected before any other procedures immediately upon opening up of the female reproductive tract by the speculum.
  • each cervical swab sample was collected from a fixed position on the peripheral side (the 12 o’clock position facing the clinician) of the external os.
  • each swab was collected by rotating 360 degrees once.
  • the swabs were collected without touching the cervical mucus plug and were sterile (DACRON swabs) .
  • DACRON swabs sterile
  • another negative control swab was collected in parallel with each cervical swab but without touching the patients.
  • the cervical swab and the negative control swabs were immersed in sterile and nuclease-free water and stored at -80°C until extraction.
  • the swabs were extracted for genomic DNA using an established method (Method B in the cited publication) (Yuan et al. 2012, PLoS One 7 (3) : e33865) , which would ensure fair representation of bacterial communities commonly found in the female reproductive tracts.
  • This method involved enzyme digestion (lysozyme, Sigma) and a column-based DNA extraction method (QiaAmp DNA extraction kit, Qiagen) . To minimize any batch variation, all samples were extracted on the same day.
  • PCR amplification and massively parallel sequencing MPS
  • the cervical swab samples inevitably would comprise human genomic DNA among the bacterial genomic DNA
  • 16S rRNA gene which is commonly possessed by all bacteria, but not by human.
  • V4 and V5 were complementary to the highly conserved regions 16S rRNA gene (Claesson et al., 2010, Nucleic Acids Res 38 (22) : e200) .
  • PCR was performed as a 50-L reaction with 2.5 units of the FastStart Taq DNA polymerase (FastStart HiFi PCR System dNTPack, Roche) , 4 mM MgCl 2 , 100 nM of each primer and 200 ⁇ M dNTPs. All PCR were run on a PTC-100 thermal cycler (Bio-Rad) using the following thermocycling conditions: 95°C for 2 minutes, followed by 33 cycles of 95°C for 30 seconds, 40°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 5 minutes and 25°C for 5 minutes. We then subjected the PCR product to electrophoresis.
  • FastStart HiFi PCR System dNTPack Roche
  • All PCR were run on a PTC-100 thermal cycler (Bio-Rad) using the following thermocycling conditions: 95°C for 2 minutes, followed by 33 cycles of 95°C for 30 seconds, 40°C for 30 seconds, and 72°
  • NBR ratio
  • SLS cumulative-sum scaling
  • each OTU was aligned against the 16S ribosomal RNA database of the GenBank (NCBI) using the BLAST algorithm. Where appropriate, species information for a given OTU was derived from the database match with the highest alignment score (i.e. the nearest match) . However, it is important to note that the taxonomic classification (i.e. kingdom, phylum, class, order, family, and genus) provided by the nearest match from BLAST or the Bayesian RDP Classifier are inherently limited by the respective databases at the GenBank and RDP.
  • the taxonomic classification i.e. kingdom, phylum, class, order, family, and genus
  • taxon and four taxa were identified as significantly increased and decreased, respectively, in the “sPTB after cerclage” group, compared with the “TB after cerclage” group (Tmisd, p ⁇ 0.05 and q ⁇ 0.05) . Thirteen taxa and four taxa were identified as significantly increased and decreased, respectively, in the “sPTB after cerclage” group, compared with the “TB after cerclage” group (Tmssd, p ⁇ 0.05 and q ⁇ 0.05) .
  • taxa and one taxon were identified as significantly increased and decreased, respectively, in the “sPTB after cerclage” group, compared with the “TB after cerclage” group (Tuv, p ⁇ 0.05 and q ⁇ 0.05) .
  • No taxon and three taxa were identified as significantly increased and decreased, respectively, in the “sPTB after cerclage” group, compared with the “TB after cerclage” group (Tev, p ⁇ 0.05 and q ⁇ 0.05) .
  • Thirteen taxa and four taxa were identified as significantly increased and decreased, respectively, in the “sPTB after cerclage” group, compared with the “TB after cerclage” group (Tmssd, p ⁇ 0.05 and q ⁇ 0.05) .
  • LRA (OTU #1, Figure 1A) , LRA (1 iOTU) -LRA (5 dOTUs) ( Figure 1C) , and LRA (13 iOTUs) - (1 dOTUs) ( Figure 1E) , which are defined in the respective figure legend are potential useful tests to identify these two sub-groups of women among the CI patients.
  • the level of any taxon measured as absolute abundance or relative abundance can be used for calculation.
  • the levels of selected taxa in a first group (Group A) can be combined in linear scale or logarithmic scale as a total level (sum A) .
  • the level of other selected taxa in a second group (Group B) can be calculated (sum B) .
  • the difference between, or ratio of sum A and sum B can be used for prediction.
  • the sum or product of sum A and sum B may also be used.
  • the abundance of each taxon was determined by 16S rRNA-based massively parallel sequencing.
  • qPCR quantitative PCR
  • Relative abundance of that taxon can be calculated by dividing the abundance of that taxon by the total bacterial load, which can be achieved by qPCR targeting universally for all bacteria in a sample.
  • CI patients positively identified with tests built on Tables 2, 3 and 4 have increased risk of sPTB ⁇ 34 weeks, PTB ⁇ 34 weeks and short latency interval ⁇ 28 days after the cerclage intervention.
  • CI patients positively identified with tests built on Tables 7, 8 and 9 have increased risk of sPTB, PTB, and/or short latency interval Thus, they are not likely to be benefit from cerclage, and should be spared from this intervention.
  • CI patients who are not tested positive by those tests are likely to benefit from cerclage, and should be offered the intervention as a measure to prevent preterm birth.
  • the 39 taxa described herein are defined by the genomic sequence of the 16S rRNA gene.
  • a database match is always limited by the completeness and coverage of the database. In particular, any matches with the percentage of nucleotide identity ⁇ 97% may imply that the concerned OTU is a previously unreported and hence novel bacterium.
  • This example describes a list of differentially abundant bacterial taxa (bacterial markers) in the cervical swab samples of CI patients undergoing intervention and resulting in preterm birth, compared with those undergoing the same intervention and resulting in term birth.
  • bacterial markers differentially abundant bacterial taxa
  • other similar lists in the literature were often obtained by testing the amniotic fluid procured by invasive procedure (e.g., amniocentesis) .
  • this method is relatively non-invasive, since cervical swab could be obtained in a minimally invasive way.
  • the method can be performed are during the second-trimester, pre-delivery stage cervical swab samples.
  • other relevant methods involve markers in the fetal membranes and placentas, which are available only after delivery.
  • the method described herein is applicable to an earlier stage of pregnancy and more useful for early detection and prevention of preterm birth.
  • the methods since ascending infection via the cervical canal is the major route of intraamniotic infection (IAI) , the methods, if applied soon enough, may detect the targeted bacteria before they appear in the intraamniotic cavity, which is an advanced and serious stage of infection.
  • the methods provided herein are useful for improving the outcome of intervention on CI patients.
  • the pre-intervention test to select patients for surgical cerclage or the pessary ring involves amniotic culture, is not only invasive, but also insensitive.
  • pre-intervention test selection of patients for the intervention are often not performed in the clinical practices, and the intervention is mostly performed blindly without an accurate diagnosis of IAI.
  • the present invention provides a highly sensitive and specific method to identify CI patients who should be more accurately ruled out before the intervention.
  • OTU operational taxonomic: unit.
  • LRA log 1o relative abunance for NBR and SUB normalization,log 2 abundance for CSS normalization (see text for NBR, SUB and CSS) . standard deviation.
  • Tuv T-test presuming unequal veriance.
  • Tev T-test presuming equal variance,Tmisd, multiple T-test (Prism 6.01) presuming independent SD of LRA for each OTU.
  • Tmssd multiple T-test presuming same SD of LRA for all OTUs.
  • p-values and q-values ⁇ 0.0001 are shown in scientific notation, where 4.56E-07 represents 4.56 ⁇ 10 -7 .
  • OTU operational taxonomic unit.
  • LAV log abundance value (log 10 relative abunance for NBR and SUB normalization, log 2 abundance for CSS normalization, see text for details) .
  • SD standard deviation. Direction of change, change in the short latency group relative to the long latency group.
  • Tuv T-test presuming unequal variance. Tev, T-test presuming equal variance.
  • Tmisd multiple T-test (Prism 6.01) presuming independent SD of LAV for each OTU.
  • Tmssd multiple T-test presuming same SD of LAV for all OTUs.
  • p-values and q-values ⁇ 0.00001 are shown in scientific notation, where 4.56E-07 represents 4.56 x 10 -7 .
  • Table 5 Features of taxa that are differentially abundant in the cervices of cervical insufficiency women resulting in spontaneous preterm birth (sPTB) /preterm birth (PTB) /short latency ⁇ 28 days after the cerclage intervention. Each taxon is specified by the genomic sequence of the 16S rRNA gene.
  • taxa that are differentially abundant in the cervices of cervical insufficiency women resulting in spontaneous preterm birth (sPTB) /preterm birth (PTB) /short latency ⁇ 28 days after the clinical intervention of placement of cerclage or pessary ring.
  • sPTB spontaneous preterm birth
  • PTB preterm birth
  • Short latency ⁇ 28 days after the clinical intervention of placement of cerclage or pessary ring.
  • Each taxon is specified by the genomic sequence of the 16S rRNA gene.
  • Cervical insufficiency a risk of preterm birth
  • PTB Preterm birth
  • RDS respiratory distress syndrome
  • BPD bronchopulmonary dysplasia
  • IVH intraventricular hemorrhage
  • PTB can be divided into 3 major categories: spontaneous preterm birth (sPTB) , iatrogenic preterm birth (iPTB) caused by pre-eclampsia and fetal growth restriction, and multiple pregnancy-related PTB.
  • sPTB spontaneous preterm birth
  • iPTB iatrogenic preterm birth
  • PTB multiple pregnancy-related PTB.
  • CI cervical insufficiency
  • ACD advanced cervical dilation
  • a shortened or dilated cervix may expose the chorioamnionic membranes to bacteria in the lower genital tract and create the conditions for an ascending infection into the amniotic cavity (intra-amniotic infection, IAI) , which greatly increases the risk for PTB.
  • IAI intra-amniotic infection
  • the CI patient may be offered surgical cerclage, which involves suturing within and around the perimeter of the cervix to keep it closed (Shirodkar, Antiseptic, 1955; 52 (2) : 299–300) .
  • the ultimate goal of cerclage treatment is to prolong the pregnancy and reduce PTB.
  • cerclage treatment is not suitable for every CI patient.
  • CI patients with IAI as detected by positive culture of amniotic fluid, often result in poor cerclage outcomes, including higher rates of PTB ⁇ 34 weeks, rupture of membrane or even neonatal death (Romero et al., Am J Obstet Gynecol, 1992, 167 (4 Pt 1) : 1086-91; Mays et al., Obstet Gynecol, 2000, 95 (5) : 652-5) . Therefore, the potential benefit of cerclage treatment in CI patients with IAI may not outweigh the surgical risk.
  • IAI is highly prevalent (38% -51% ) in CI patients (Romero et al., Am J Obstet Gynecol, 1992, 167 (4 Pt 1) : 1086-91; Mays et al., Obstet Gynecol, 2000, 95 (5) : 652-5)
  • experts have suggested to rule out IAI using pre-cerclage amniocentesis to detect for microorganisms (Berghella et al., Am J Obstet Gynecol, 2013, 209 (3) : 181-92; Airoldi et al., Am J Perinatol, 2009, 26 (1) : 63-8) . This may spare patients who are unlikely to benefit from cerclage treatment from its surgical risks.
  • rRNA fungal/bacterial ribosomal RNA
  • a logistic regression was performed to ascertain the effects of LA7 value and treatment type (cerclage/pessary) on the likelihood that participants have poor treatment outcome (i.e., “sPTB after treatment” ) .
  • the median values of LA7 were 3.35 and 0.845 in the “sPTB after treatment” and the “TB after treatment” groups, respectively ( Figure 8A) .
  • the median LA7 values are shown to be increased by 3.96-fold in the former group (Mann-Whitney, p ⁇ 0.0001) .
  • a multiple pregnancy ⁇ 2 fetuses
  • uterine abnormality e.g., myoma, ASCUS
  • Cervical swab collection Before the cerclage treatment, a cervical swab sample was collected from the CI patient by rotating a sterile Dacron swab 360° once on the peripheral side (the 12 o’clock position facing the clinician) of the external os. This was performed immediately upon opening up of the reproductive tract by speculum.
  • Raw reads were de-multiplexed, denoised, quality-filtered and analyzed with settings similar to Cheung et al., PLoS One, 2013, 8 (1) : e54574.
  • Quality-filtered reads were de-duplicated and clustered into Otu at 97% similarity using the mothur program suite (Schloss et al., Appl Environ Microbiol, 2009, 75 (23) : 7537-41) .
  • Each Otu was be taxonomically classified by matching against the latest Ribosomal Project Database or the NCBI 16S rRNA database, and calculated for its read count per sample. Total read count was calculated by summing up the read counts of all Otu identified in each sample.
  • the CSS normalization does not require the estimation of the amount of total bacterial genomic DNA or human genomic DNA (e.g., the ⁇ -actin, ⁇ -globin, GAPDH genes) in the clinical sample. Hence, the CSS-normalised abundance values were unaffected by how hard/gentle the swab sample is obtained from the patient.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode de prédiction du risque d'une issue défavorable de grossesse (ex. naissance avant terme) pour un sujet enceinte, par détection du niveau élevé ou réduit de bactéries provenant d'un ou de plusieurs taxons bactériens sélectionnés (par ex. genres ou espèces). L'invention concerne également un kit utile à une telle méthode. En outre, l'invention concerne une méthode pour déterminer le risque de présenter une dilatation du col de l'utérus avancée et/ou un raccourcissement du col de l'utérus prématuré sur la base de la différence d'abondance de taxons bactériens. Par ailleurs, la présente invention concerne des méthodes pour prédire si un sujet enceinte bénéficiera ou non d'une intervention clinique pour empêcher la naissance avant terme.
PCT/CN2015/097341 2014-12-15 2015-12-15 Détection de taxons bactériens pour la prédiction d'une naissance avant terme après une intervention clinique Ceased WO2016095789A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462092128P 2014-12-15 2014-12-15
US62/092,128 2014-12-15

Publications (1)

Publication Number Publication Date
WO2016095789A1 true WO2016095789A1 (fr) 2016-06-23

Family

ID=56125931

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/097341 Ceased WO2016095789A1 (fr) 2014-12-15 2015-12-15 Détection de taxons bactériens pour la prédiction d'une naissance avant terme après une intervention clinique

Country Status (1)

Country Link
WO (1) WO2016095789A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170362640A1 (en) * 2016-06-16 2017-12-21 Life Technologies Corporation Novel compositions, methods and kits for microorganism detection
WO2019100113A1 (fr) 2017-11-24 2019-05-31 The University Of Western Australia Méthode de diagnostic d'accouchement prématuré associé à une infection
CN110582582A (zh) * 2017-09-04 2019-12-17 梨花女子大学校产学协力团 根据利用样本中微生物群落变化的早产风险预测
US10774377B1 (en) 2017-10-05 2020-09-15 Verily Life Sciences Llc Use of unique molecular identifiers for improved sequencing of taxonomically relevant genes
RU2793917C2 (ru) * 2017-11-24 2023-04-10 Зе Юниверсити Оф Уэстерн Острейлиа Способ диагностики связанных с инфекцией преждевременных родов
FR3151603A1 (fr) * 2023-07-27 2025-01-31 Universite Clermont Auvergne Procédé de détection d’une pathologie

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009094665A1 (fr) * 2008-01-25 2009-07-30 Perkinelmer Health Sciences, Inc. Procédés de détermination du risque de complications prénatales
WO2011053666A1 (fr) * 2009-10-29 2011-05-05 The Trustees Of The University Of Pennsylvania Procédé de prédiction de risque de naissance prématurée
CN101063677B (zh) * 2006-04-30 2012-07-25 安徽省生物医学研究所 一种预测妊娠不良结局发生风险的试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101063677B (zh) * 2006-04-30 2012-07-25 安徽省生物医学研究所 一种预测妊娠不良结局发生风险的试剂盒
WO2009094665A1 (fr) * 2008-01-25 2009-07-30 Perkinelmer Health Sciences, Inc. Procédés de détermination du risque de complications prénatales
WO2011053666A1 (fr) * 2009-10-29 2011-05-05 The Trustees Of The University Of Pennsylvania Procédé de prédiction de risque de naissance prématurée

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170362640A1 (en) * 2016-06-16 2017-12-21 Life Technologies Corporation Novel compositions, methods and kits for microorganism detection
US12054790B2 (en) 2016-06-16 2024-08-06 Life Technologies Corporation Compositions, methods and kits for microorganism detection
CN110582582A (zh) * 2017-09-04 2019-12-17 梨花女子大学校产学协力团 根据利用样本中微生物群落变化的早产风险预测
EP3680351A4 (fr) * 2017-09-04 2021-06-09 Ewha University-Industry Collaboration Foundation Prédiction de risque de naissance prématurée à l'aide d'un changement de communauté microbienne dans un échantillon
US10774377B1 (en) 2017-10-05 2020-09-15 Verily Life Sciences Llc Use of unique molecular identifiers for improved sequencing of taxonomically relevant genes
WO2019100113A1 (fr) 2017-11-24 2019-05-31 The University Of Western Australia Méthode de diagnostic d'accouchement prématuré associé à une infection
JP2021514611A (ja) * 2017-11-24 2021-06-17 ジ ユニバーシティ オブ ウェスタン オーストラリア 感染症関連早産診断方法
RU2793917C2 (ru) * 2017-11-24 2023-04-10 Зе Юниверсити Оф Уэстерн Острейлиа Способ диагностики связанных с инфекцией преждевременных родов
AU2018373494B2 (en) * 2017-11-24 2024-06-06 The University Of Western Australia Infection-related preterm birth diagnostic method
FR3151603A1 (fr) * 2023-07-27 2025-01-31 Universite Clermont Auvergne Procédé de détection d’une pathologie

Similar Documents

Publication Publication Date Title
KR102207858B1 (ko) 유해 임신 결과를 예측하기 위한 박테리아 분류군의 검출
US10947593B2 (en) Biomarkers for premature birth
JP5789605B2 (ja) 染色体異数性の検出方法
CN110291210B (zh) 创伤性脑损伤的生物标志物
JP7000658B2 (ja) 肝臓病変を評価する方法
WO2016095789A1 (fr) Détection de taxons bactériens pour la prédiction d'une naissance avant terme après une intervention clinique
CN101260429A (zh) 作为妊娠相关病症的诊断标志物的循环mRNA
Lambertini et al. A sensitive functional assay reveals frequent loss of genomic imprinting in human placenta
EP4189117B1 (fr) Utilisation d'arn circulaire pour le diagnostic de sclérose multiple
US20240294985A1 (en) Preeclampsia diagnosis
CN111933216B (zh) 肠道微生物作为子痫前期生物标志物的用途
CN116622842A (zh) 一种用于肺腺癌非创早期诊断、药效评估的分子标志物及试剂盒
KR102845327B1 (ko) 임신부 질액의 마이크로바이옴 분석을 이용한 조산 예측용 조성물 및 조산 예측 방법
US20200340036A1 (en) Dna sequences related to diagnosis and treatment of systemic inflammatory response syndrome
WO2025011401A1 (fr) Marqueurs d'arn circulant à usage diagnostique
WO2025055728A1 (fr) Marqueurs circulants pour la parturition
CN113943810A (zh) 子宫内膜癌检测的试剂及试剂盒
CN111733265A (zh) 与复发性流产相关的阴道菌群及其应用
WO2025078830A1 (fr) Septicémie néonatale
JP2024161911A (ja) アトピー性皮膚炎の重症度の検出方法
WO2022186297A1 (fr) Procédé de détection de l'exacerbation du prurit due à la dermatite atopique
WO2022186296A1 (fr) Procédé de détection de la détérioration de la gravité de la dermatite atopique
WO2021112237A1 (fr) Procédé de prédiction de l'apparition de la chorioamnionite
CN110643700A (zh) Kfs相关基因突变在制备检测试剂盒中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15869296

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15869296

Country of ref document: EP

Kind code of ref document: A1