[go: up one dir, main page]

WO2025055728A1 - Marqueurs circulants pour la parturition - Google Patents

Marqueurs circulants pour la parturition Download PDF

Info

Publication number
WO2025055728A1
WO2025055728A1 PCT/CN2024/115026 CN2024115026W WO2025055728A1 WO 2025055728 A1 WO2025055728 A1 WO 2025055728A1 CN 2024115026 W CN2024115026 W CN 2024115026W WO 2025055728 A1 WO2025055728 A1 WO 2025055728A1
Authority
WO
WIPO (PCT)
Prior art keywords
labor
woman
blood
mrna
onset
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.)
Pending
Application number
PCT/CN2024/115026
Other languages
English (en)
Inventor
Tak Yeung Leung
Ka Wing Karen WONG
Yee Ling KO
Wenjing DING
Yik Lok CHUNG
Joyce Ka Yu TSE
Ting Fung Philos CHAN
Chi Chiu Wang
Siu Chung Stephen Chim
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 WO2025055728A1 publication Critical patent/WO2025055728A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • IOL induction of labor
  • cfRNA cell-free RNA
  • This method requires measuring the cfRNA levels of nine genes and taking multiple blood samples from each pregnant woman, however, sophisticated detection platform (e.g., multiplex PCR, RNA-seq) and multiple hospital visits are therefore required and present challenges in practicing this method.
  • the present inventors provide a new method for predicting delivery using the RNA level of only one gene and only one maternal blood sample per woman.
  • the invention is based on the systematic identification of delivery-associated circulating RNA transcripts (DACTs) , the levels of which in peripheral blood were changed before and after the latent phase of labor in women who underwent a successful IOL. It has been illustrated that maternal blood level of a DACT before IOL is useful for the prediction of successful IOL, ending in spontaneous delivery. Thus, women predicted by the DACT test to have a low chance of successful IOL may choose to give birth directly by Cesarean delivery rather than undergoing the potentially unnecessary pain of induced labor. Further, DACTs are useful for the prediction of spontaneous labor or spontaneous preterm birth, which precedes preterm labor, where regulation of some circulating markers in common to induced labor may be involved.
  • DACTs delivery-associated circulating RNA transcripts
  • RNA markers circulating in the blood stream of a pregnant woman for the purpose of predicting the timing of onset of labor and child delivery, therefore assessing whether induction of labor should be performed for the pregnant woman.
  • the present inventors have identified several new circulating mRNA markers, each of which can be used alone in as few as one single blood sample taken from a pregnant woman to achieve high performance prediction of onset of labor, which in turn allows for determination of what type of medical intervention may be appropriate for individual pregnant women.
  • the present invention provides a method for assessing mRNA level.
  • the method includes these steps: (a) obtaining a blood, plasma, or serum sample from a pregnant woman prior to onset of labor, optionally further obtaining another blood, plasma, or serum sample from the woman during latent phase of labor; and (b) quantifying mRNA level of only one of GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 in the blood, plasma, or serum sample (s) from step (a) , but no other blood, plasma, or serum sample is taken from the woman.
  • only one of GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA level is determined in the sample (s) , but no other mRNA level in the sample (s) is quantified.
  • the level or levels of one or two or more reference marker level in the sample (s) are quantified and are used for normalizing the level of one of GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA, but no other mRNA level in the sample (s) is quantified.
  • only one blood, plasma, or serum sample is obtained prior to onset of labor.
  • only one of GMEB2, PDCD11, IGLV1-36, UPF2, or TRBV15 mRNA level is quantified.
  • the onset of labor in the pregnant woman is predicted as within about t hours from step (a) , with s and t listed in Tables 11A (1) and 11D (1) , respectively.
  • onset of the active phase of labor in the pregnant woman is predicted as within a median of 3.70 hours after blood-sampling (Fig. 2A, GMEB2, green curve) .
  • onset of the active phase of labor is predicted as within a median of 14.14 hours after blood-sampling (Fig. 2A, GMEB2, red curve) .
  • the method may further comprises, after step (b) , a step of performing induction of labor (IOL) on the pregnant woman, for example, if the woman is predicted to begin labor within about 2-5 hours.
  • steps (a) and (b) are performed on two pregnant women, among whom one woman has a higher level of GMEB2, PDCD11, IGLV1-36, UPF2, or TRBV15 mRNA and one woman has a lower level of the same mRNA.
  • the method may further comprise, after step (b) , performing an induction of labor on the pregnant woman with the lower mRNA level and performing a Cesarean section on the pregnant woman with the higher mRNA level.
  • TRAPPC11 only one of TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA level is quantified.
  • the onset of labor in the pregnant woman is predicted as within about t hours from step (a) , with s and t listed in Tables 11A (1) and 11C (1) , respectively.
  • the maternal blood mRNA level of TRAPPC11 is higher than the 65th percentile of a population of third-trimester women, then onset of the active phase of labor in the pregnant woman is predicted as within a median of 8.83 hours after blood-sampling (Fig.
  • the method may further comprise, after step (b) , a step of performing an induction of labor on the pregnant woman, if she is deemed to likely begin labor within about 2-5 hours.
  • steps (a) and (b) are performed on two pregnant women, among whom one woman has a higher level of TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA and one woman has a lower level of the same mRNA.
  • the method further comprises, after step (b) , performing an induction of labor on the pregnant woman with the higher mRNA level and performing a Cesarean section on the pregnant woman with the lower mRNA level.
  • a first blood, plasma, or serum sample is obtained from the woman prior to onset of labor and a second blood, plasma, or serum sample is obtained from the woman during latent phase of labor, and wherein a Rate of Change (RoC) is calculated as follows:
  • only one of GMEB2, PDCD11, IGLV1-36, UPF2, or TRBV15 mRNA level is quantified in both the first and second samples.
  • the RoC when the RoC is lower than a standard control value corresponding to s percentile of a control group, child delivery by the pregnant woman is predicted as within about t hours from the time when the first sample is obtained, where s and t are listed in Tables 11A and D, respectively.
  • the RoC of maternal blood mRNA level of GMEB2 is lower than the 20th percentile of a population of third-trimester women, then child delivery by the pregnant woman is predicted as within a median of 9.82 hours after blood-sampling (Fig.
  • the method further comprises, after step (b) , a step of performing an induction of labor on the pregnant woman, for example, if the woman is predicted to deliver her child within about 2-5 hours.
  • steps (a) and (b) are performed on two pregnant women for the same mRNA, i.e., one of GMEB2, PDCD11, IGLV1-36, UPF2, or TRBV15 mRNA, among whom one woman has a higher RoC and one woman has a lower RoC.
  • the method further comprises, after step (b) , performing an induction of labor on the pregnant woman with the lower RoC and performing a Cesarean section on the pregnant woman with the higher RoC.
  • TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA level is quantified in both the first and second samples.
  • RoC when the RoC is higher than a standard control value corresponding to s percentile of a control group, child delivery by the pregnant woman is predicted as within about t hours from the time when the first sample is obtained, where s and t are listed in Tables 11A and C, respectively.
  • the RoC of maternal blood mRNA level of TRAPPC11 is higher than the 65th percentile of a population of third-trimester women, then child delivery by the pregnant woman is predicted as within a median of 11.15 hours after blood-sampling (Fig.
  • the method further comprises, after step (b) , a step of performing an induction of labor on the pregnant woman, for example, if child delivery is predicted within about 2-5 hours.
  • steps (a) and (b) are performed on two pregnant women for the same mRNA, i.e., one of TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA, among whom one woman has a higher RoC and one woman has a lower RoC.
  • the method further comprises, after step (b) , performing an induction of labor on the pregnant woman with the higher RoC and performing a Cesarean section on the pregnant woman with the lower RoC.
  • the mRNA level is quantified by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) .
  • the present invention provides a kit for assessing mRNA level.
  • the kit includes (1) a first container containing a reagent for quantifying GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA in a blood, plasma, or serum sample; (2) a standard control for GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 mRNA level in the blood, plasma, or serum sample.
  • the kit comprises two or more of reagents for quantifying mRNA level by RT-qPCR. In some embodiments, the kit further comprises an instruction manual providing instructions for the proper usage of the kit.
  • the present invention provides uses of newly identified circulating mRNA markers, namely GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, and PADI4, in a blood sample taken from a pregnant woman, for the purpose of predicting the timing of onset of labor and child birth in the pregnant woman.
  • these uses are detailed in the methods described above and herein.
  • Figure 1 Maternal blood transcriptomes before labor and during the latent phase of labor.
  • A RNA-seq was performed on two serially-collected whole blood samples from each woman. Differentially expressed transcripts are identified (Wilcoxon signed rank test, False Discovery Rate (FDR) -adjusted p ⁇ 0.05) . Down-regulated (lower panel) and up-regulated (upper) transcripts during the latent phase. RNA levels of transcripts (rows) in women (columns) are shown as Z-score per row (red: high; grey: low) . The bar graph shows the time (hours) between blood collection and delivery.
  • FDR False Discovery Rate
  • RNA-seq-identified differentially expressed transcripts using reverse-transcriptase qPCR on an independent cohort. Transcripts expected to be down- (left) or up-regulated (right) during labor as suggested by the RNA-seq experiment are subjected to qPCR validation. RNA levels of target transcripts are normalized by geometric means of those of two reference genes, CNOT and EYA3. Asterisk indicates Wilcoxon signed rank test, p ⁇ 0.05.
  • Figure 2 Kaplan Meier analyses. Women were categorized according to high (red curves) or low (green curve) levels or rate of change (RoC) of transcripts in maternal blood using the threshold specified in Tables 11A and B. *, Logrank test p ⁇ 0.05.
  • A, B The levels of transcripts in the 1 st (pre-labor) maternal blood sample and the time between its collection and onset of active phase or delivery, respectively.
  • C, D The levels of transcripts in the 2 nd (latent-phase) maternal blood sample and the time between its collection and onset of active phase or delivery, respectively.
  • Figure 3 Pre-labor maternal blood levels of transcripts identified in the RNA-seq study and timing of onset of active phase or delivery. Key data from (A-C) down-regulated (NOMO1, TBRV15, LINC01857 mRNA transcripts) or (D-F) up-regulated (CASP4, LTB4R and MBOAT1 mRNA transcripts) during latent phase of labor.
  • Figure 4 Cross-validation on predictive performance of RT-qPCR assay of PDCD11 transcript.
  • Fig. 4A Prediction of onset of active phase within 10 hours after blood sampling (100-fold, mean sensitivity 0.870, mean specificity 0.795, mean accuracy 0.812) .
  • Fig. 4B Prediction of delivery within 10 hours after blood sampling (100-fold, mean sensitivity 0.870, mean specificity 0.795, mean accuracy 0.810) .
  • Figure 5 Gene ontology (biological processes) terms analysis of differentially expressed transcripts before onset and in latent phase of labor. Over-/under-represented biological processes with the10 greatest or lowest fold enrichment or depletion scores are shown. No fold enrichment or FDR filtering if less than 10 biological processes were identified to be associated with the input transcript lists. Differential transcripts refer to the differentially expressed (including upregulated and downregulated) transcripts.
  • FIG. 6 Performance of delivery-associated circulating transcripts (DACTs) for the prediction of vaginal delivery after IOL in the cross-sectional validation cohort.
  • DACTs delivery-associated circulating transcripts
  • Pre-labor maternal blood samples were collected from 100 pregnant women who planned to undergo IOL. Levels of DACTs were quantified and normalized. The subjects were balance-split into the train and test sets. In the train set, for each DACT, an optimal threshold in prediction of successful IOL (i.e., ending in vaginal delivery) was determined. Using this threshold, in the test set, the performance in prediction of successful IOL of each DACT was assessed, plotted (red curve) and compared to the performance in prediction of the same using cervical status based on Bishop score (grey dotted curve) .
  • 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 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, esophagus 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., tongue, colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, stomach tissue, esophagus, etc. ) among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy and may comprise colonoscopy or endoscopy. 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.
  • blood refers to a blood sample or preparation from an individual being tested for the possible presence or risk of a particular medical condition, e.g., a pregnant woman being tested for a pregnancy-associated condition or for the purpose of assessing the timing of the onset of labor and child delivery.
  • the term encompasses whole blood or any fractions of blood having varying concentrations or even no hematopoietic or any other types of cells or cellular remnants of maternal or fetal origin, including platelets.
  • blood include plasma and serum.
  • a blood sample that is essentially free of cells is also referred to as "acellular, " where no detectable quantity of blood cells is present.
  • nucleic acid or “polynucleotide” 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 is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons) .
  • an “increase” or a “decrease” refers to a detectable positive or negative change in quantity from a comparison value, e.g., an established standard control.
  • 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.
  • Other terms indicating quantitative changes or differences from a comparative basis, such as “more, “ “less, " “higher, “ and “lower, " are used in this application in the same fashion as described above.
  • the term “substantially the same” or “substantially lack of change” indicates little to no change in quantity from the standard control value, typically within ⁇ 10%of the standard control, or within ⁇ 5%, 2%, or even less variation from the standard control.
  • Standard control refers to a predetermined amount or concentration of a polynucleotide sequence or polypeptide, e.g., the DNA, RNA or protein of a pre-determined marker gene, that is present in an established normal disease-free tissue sample, e.g., a blood sample taken from a healthy pregnant woman with a healthy fetus with normal onset time for labor and delivery.
  • 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 DNA, RNA or protein of the marker gene that is present in a test sample.
  • An established sample serving as a standard control provides an average amount of the marker DNA, RNA or protein that is typical for a particular type of biological sample of an average, healthy human without a specific disease or condition of interest as conventionally defined (e.g., preterm labor or preterm birth among pregnant women) .
  • a standard control value may vary depending on the nature of the sample as well as other factors such as the gender, age, ethnicity of the subjects based on whom such a control value is established.
  • the standard control information may also be expressed in the form of a range of values, each corresponding to a percentile between the low end (0 percentile) to the high end (100 percentile) of the marker amount/level found in the healthy control subjects.
  • average refers to certain characteristics, especially the amount of a biomarker DNA, RNA or protein, found in the person's biological sample, that are representative of a randomly selected group of healthy humans who are free of the disease (such as preterm labor or preterm birth among pregnant women) .
  • This selected group should comprise a sufficient number of humans such that the average amount of the marker DNA, RNA or protein in the type of sample among these individuals reflects, with reasonable accuracy, the corresponding amount of marker DNA, RNA, or protein in the general population of healthy humans.
  • other factors such as gender, 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 polynucleotide of interest or a polypeptide of interest, e.g., a pre-selected human biomarker in its DNA, RNA or protein form, present in a sample. Such quantity may be expressed in the absolute terms, i.e., the total quantity of the polynucleotide or polypeptide in the sample, or in the relative terms, i.e., the concentration of the polynucleotide or polypeptide in the sample.
  • treat or “treating, " as used in this application, describes to an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a relevant condition.
  • treating a condition encompasses both therapeutic and prophylactic intervention against the condition.
  • an effective amount of an therapeutic agent for treating or preventing a particular disease or condition is the amount of said therapeutic to achieve a detectable effect on the condition, such that the symptoms of condition are reduced, reversed, eliminated, prevented, or delayed of the onset in a patient who has been given the agent for therapeutic purposes.
  • An amount adequate to accomplish the specific purpose is defined as the “prophylactically effective dose” or “therapeutically effective dose. " The dosing range varies with the nature of the therapeutic agent being administered and other factors such as the route of administration and the severity of a patient’s condition.
  • subject or “subject in need of treatment, " as used herein, includes individuals who seek medical attention due to risk of, or actual suffering from, a pre-determined disease or condition, such as a pregnancy-associated disorder (e.g., preterm labor or preterm birth among pregnant women) .
  • Subjects also include individuals currently undergoing therapy that seek manipulation of the therapeutic regimen.
  • Subjects or individuals in need of treatment include those that demonstrate symptoms of the condition or are at an increased risk of suffering from this condition or its symptoms.
  • a subject in need of treatment includes individuals with a genetic predisposition or family history for any one of pregnancy-associated disorder (such as preterm labor or preterm birth) , those that have suffered relevant symptoms in the past, those that have been exposed to a triggering substance or event, as well as those suffering from chronic or acute symptoms of the condition.
  • a “subject in need of treatment” may be at any age of life.
  • the present invention provides new and improved methods for predicting IOL outcome by assessing the level of as few as one mRNA transcript in the circulating maternal blood in as few as one single blood sample taken from a pregnant woman prior to her onset of labor.
  • routine clinical method such as Bishop score
  • the method disclosed herein was able to achieve improved performance with a higher area under ROC curve (AUC) .
  • AUC area under ROC curve
  • the method of this invention is much simpler in operation in that it requires no specialized ultrasound equipment or trained/certified operators.
  • the method of this invention is easy to operate and does not require sophisticated detection platform. Also in contrast to the existing methods requiring multiple blood draws from each pregnant woman, the method of this invention requires as few as only one blood sample from each woman. Furthermore, the existing methods require detection of multiple biomarkers by sophisticated instrument, whereas this method requires the measurement of only one circulating RNA marker for the prediction of a successful IOL. Thus, the method of this invention presents the significant advantage in its adaptability as an easy point-of-care test, allowing healthcare professionals to rapidly gain necessary information in order to choose the most appropriate intervention in perinatal care. None of the existing methods are capable of such.
  • 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
  • sequence of interest used in this invention e.g., the polynucleotide sequence of the human GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, or PADI4 gene, and synthetic oligonucleotides (e.g., primers) can be verified using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene 16: 21-26 (1981) .
  • the present invention relates to measuring the amount of an analyte, or a biomarker of diagnostic value, such as a DNA, an RNA (e.g., mRNA) , a protein, or a molecule of another chemical nature, found in a biological sample, especially a fluid sample such as a sample of a bodily fluid (e.g., blood or any fraction thereof) , secretion, perspiration, or excretion, as a means to detect the presence, to assess the risk of developing, and/or to monitor the progression or treatment efficacy of a pre-selected disease or condition (such as a pregnancy-associated condition, for example, preterm labor or preterm birth among pregnant women) .
  • a pre-selected disease or condition such as a pregnancy-associated condition, for example, preterm labor or preterm birth among pregnant women.
  • the first steps of practicing this invention are to obtain an appropriate biological sample from a test subject and extract the analyte, e.g., mRNA or DNA or protein,
  • An appropriate biological sample is obtained from a person to be tested or monitored for a pertinent disease or condition (e.g., preterm labor or preterm birth among pregnant women) using a method of the present invention. Collection of a tissue sample or a blood sample from an individual is performed in accordance with the standard protocol hospitals or clinics generally follow, such as during a biopsy or blood draw process. An appropriate amount of tissue or blood or any other fluid is collected and may be stored according to standard procedures prior to further preparation.
  • a pertinent disease or condition e.g., preterm labor or preterm birth among pregnant women
  • RNA or DNA analyte found in a subject's tissue or blood sample may be performed using, e.g., an acellular fraction of whole blood such as plasma or serum.
  • an acellular fraction of whole blood such as plasma or serum.
  • the methods for preparing biological samples for nucleic acid extraction are well known among those of skill in the art.
  • a subject's tissue or blood sample should be first treated to disrupt cellular membrane so as to release nucleic acids contained within the cells.
  • the step of disruption of cell membrane is not needed in the case of an acellular blood fraction (e.g., plasma or serum) is used.
  • RNA isolation There are numerous methods for extracting RNA from a biological sample.
  • the general methods of mRNA 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 RNA from a biological sample from a test subject. Combinations of more than one of these methods may also be used.
  • RNA is extracted from a sample, the amount of a biomarker RNA may be quantified.
  • the preferred method for determining the RNA level is an amplification-based method, e.g., by polymerase chain reaction (PCR) , especially reverse transcription-polymerase chain reaction (RT-PCR) .
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • a DNA copy (cDNA) of the biomarker RNA is ususally 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
  • Methods suitable for PCR amplification of ribonucleic acids are described by Romero and Rotbart in Diagnostic Molecular Biology: Principles and Applications pp. 401-406; Persing et al., eds., Mayo Foundation, Rochester, MN, 1993; Egger et al., J. Clin. Microbiol. 33: 1442-1447, 1995; and U.S. Patent No. 5,075,212.
  • 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.
  • 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 mRNA 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.
  • the selected group of healthy individuals must be of a reasonable size, such that the average amount/concentration of the biomarker in the pertinent tissue or fluid sample obtained from the group can be reasonably regarded as representative of the normal or average level of the biomarker in the same type of samples among the general population of healthy people.
  • the selected group comprises at least 10 human subjects.
  • a much larger population of healthy individuals e.g., at least 50, 100, 200, or more individuals
  • serve as a control group serve as a control group to provide standard control value, which may be represented in a value range rather than only a single average or mean value.
  • the values within this range represent percentiles from 0 to 100, reflecting their ranks among the entire control group from the lowest to the highest in a biomarker’s amount/concentration.
  • this average or median or representative value or profile is considered a standard control.
  • a standard deviation is also determined during the same process.
  • separate standard controls may be established for separately defined groups having distinct characteristics such as age, gender, or ethnic background.
  • the same normalization step is also carried out in the generation of the corresponding standard control value.
  • the present invention further provides a means for a healthcare professional to determine which medical intervention to choose in order to facilitate a pregnant woman in her successful completion of her pregnancy.
  • a maternal blood e.g., plasma or serum
  • two or more pregnant women are tested together in accordance with the methods of this invention in order to determine their relative timing of the onset of labor or active phase of child delivery and therefore their relative likelihood of success if they receive IOL.
  • a maternal blood e.g., whole blood, plasma or serum
  • a circulating mRNA selected from the group consisting of GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, and PADI4
  • IOL may be administered to the first woman, as deemed appropriate by an attending physician taking into consideration other relevant factors, but no IOL is administered to the second woman. Rather, the second woman may undergo a C-section, if deemed appropriate by an attending physician upon considering other relevant factors.
  • the invention provides compositions and kits for practicing the methods described herein to assess the level of a biomarker (e.g., a circulating mRNA of predict value for the timing of the onset of labor in a pregnant woman) in a sample taken from a subject being tested, which can be used for various purposes such as for assessing the profile of the biomarker expression in this type of samples or for assessing the timing of labor/delivery or the likelihood of successful IOL among pregnant women.
  • a biomarker e.g., a circulating mRNA of predict value for the timing of the onset of labor in a pregnant woman
  • Kits for carrying out assays for determining the biomarker level typically include at least one agent useful for the detection, especially quantitative detection, of the biomarker.
  • the kits include a plurality of reagents suitable for performing quantitative analysis of any one of the biomarkers specified in this application, namely a circulating mRNA selected from the group consisting of GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, and PADI4, such as RT-PCR, especially RT-qPCR.
  • These reagents may be placed in a single container, e.g., in the form of a pre-made reaction mixture, or may be placed in several separate containers, each containing one or more of the reagents.
  • kits also include an appropriate standard control.
  • the standard controls indicate the average value of a biomarker in the same type of sample (e.g., plasma or serum sample) from healthy subjects not suffering from a condition being tested for (e.g., healthy pregnant women not at any increased risk for preterm labor or preterm birth) .
  • a condition being tested for e.g., healthy pregnant women not at any increased risk for preterm labor or preterm birth
  • 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 test results, e.g., the timing of onset of labor or active phase of delivery, or the likelihood of a successful IOL.
  • the present invention can also be embodied in a device or a system comprising one or more such devices, which is capable of carrying out all or some of the method steps described herein.
  • the device or system performs the following steps upon receiving a biological sample, e.g., a blood (such as a plasma or serum) sample taken from a pregnant woman being tested for predicting the timing of the onset of labor, assessing the likelihood of a successful IOL: (a) determining in a blood sample taken from a first pregnant woman the amount or concentration of a biomarker, which is one circulating mRNA selected from the group consisting of GMEB2, PDCD11, IGLV1-36, UPF2, TRBV15, TRAPPC11, DHRS13, SH3BP5, CA4, and PADI4; and (b) comparing the biomarker quantity with a standard control value or with the quantity of the same biomarker in a sample of the same type taken from a second pregnant woman; and (c) providing an a biological sample, e.g.
  • the device or system of the invention performs the task of steps (b) and (c) , after step (a) has been performed and the biomarker quantity from step (a) has been entered into the device.
  • the device or system is partially or fully automated.
  • IOL induction of labor
  • C-section Cesarean section
  • the present invention provides an improved and simpler method for predicting labor and delivery by analyzing only one mRNA species and requiring only one maternal blood sample from a pregnant woman.
  • the present inventors first performed a systematic identification of delivery-associated circulating RNA transcripts or DACTs, the levels of which in peripheral blood are shown to increase or decrease before and after the latent phase of labor in women who are successfully induced.
  • DACTs delivery-associated circulating RNA transcripts
  • Each of the DACT level in maternal blood prior to induction is useful for the prediction of a successful IOL in a woman.
  • the method of this invention Compared to routine clinical method (Bishop score) , the method of this invention has achieved a higher area under ROC curve (AUC) (Fig. 6) .
  • AUC area under ROC curve
  • the method of this invention does not require ultrasound equipment, training and certification of ultrasonographers.
  • the method of this invention is easy to operate and does not require sophisticated detection platform. While the existing methods require collection of multiple blood samples from each woman, the method of this invention requires collection of only one blood sample from each woman. While the existing methods require detection of multiple biomarkers by sophisticated instrument, the method of this invention only requires the measurement of one circulating RNA marker for the prediction of parturition event (e.g., spontaneous delivery after induced labor, i.e., successful IOL) . As such, the method of this invention, but not the existing method, can be easily converted into a simple point-of-care test, which does not require the sending of samples for testing in a centralized laboratory. This is particularly relevant to perinatal care, when timely and accurate results may facilitate rapid and appropriate intervention.
  • parturition event e.g., spontaneous delivery after induced labor, i.e., successful IOL
  • IOL induction of labor
  • cfRNAs cell-free RNA transcripts
  • a model comprising 9 cfRNAs, namely that coding for CGA, CAPN6, CGB, ALPP, CSHL1, PLAC4, PSG7, PAPPA, and LGALS14, was built to estimate gestational age of a maternal blood sample.
  • cfRNAs were shown in this research setting to be useful for predicting the gestational age at delivery at comparable accuracy (45%) to ultrasound (57.8%) (Ngo et al. 2018)
  • the use of multiple markers and multiple blood samples drawn from each woman may present challenge when this method is practiced in a clinical setting.
  • the detection of multiple RNA markers usually requires multiplexed reverse-transcription quantitative PCR which relies on multiple fluorescent probes.
  • sophisticated equipment is required to implement this method, probably in a centralized laboratory.
  • RNA sequencing provided opportunities for global and deeper integration in protein-coding mRNAs with regulating signals such as non-coding RNAs and alternative RNA splice events. This technology has been applied by our team to better understand the differences in the circulating transcripts in preterm labor women ending in spontaneous preterm birth and those ending in term birth (Chim et al. 2012) , as well as the pathomechanisms of placenta in selective intrauterine growth restriction (Li et al. 2020) .
  • RNA sequencing is used in this study to systematically quantify the dynamic changes in the circulating RNA transcripts before and after the onset of labor and to dissect the underlying molecular regulation involved in the initiation of human parturition.
  • This observational study comprised three modules: (i) a longitudinal discovery study; (ii) a longitudinal validation study; and (iii) a cross-sectional validation study.
  • modules (i) and (ii) women who underwent a successful IOL (i.e., ending in vaginal/spontaneous delivery) were recruited, whereas in module (iii) women who underwent a successful IOL or an unsuccessful IOL (i.e., ending in Cesarean delivery) were both recruited.
  • RNA-seq Strand-specific RNA-sequencing
  • module (ii) the longitudinal validation study, two serial maternal blood samples were collected similarly (one before IOL and another during the latent phase) but from another 27 women. Validation of the DACTs identified in module (i) was performed using reverse-transcriptase (RT) quantitative polymerase chain reactions (qPCR) on these samples independent from the discovery phase. Relationship between the maternal blood levels of differentially expressed transcripts and the timing of onset of active phase of labor or delivery were reported. Further, the potential use of the differentially expressed transcripts in prediction of the onset of active phase or delivery within a given time period was investigated.
  • RT reverse-transcriptase
  • qPCR quantitative polymerase chain reactions
  • module (iii) the cross-sectional validation study, one sample was collected one before IOL from another cohort of women who did not participate in the discovery and the longitudinal validation study. These women were followed until delivery and their the outcomes of labor including the final mode of delivery were recorded. Women who underwent a successful IOL were matched with women who underwent an unsuccessful IOL for clinical characteristics such as parity and cervical status at blood-taking which may confound the results of the study. Levels of DACTs were measured using RT-qPCR. Performance of each DACT in the prediction of IOL outcome (i.e., vaginal delivery or Cesarean delivery) were assessed by the cross-validation approach.
  • IOL outcome i.e., vaginal delivery or Cesarean delivery
  • modules (i) to (iii) women who required IOL in the study unit, Department of Obstetrics &Gynaecology, Prince of Wales Hospital, Hong Kong were invited to participate. Before IOL, a 1.5-mL maternal peripheral whole blood sample was drawn from the recruited women into an EDTA tube and immediately preserved in 6.5 mL of RNAlater.
  • the second sample was collected during the latent phase, which was defined as presence of uterine contractions with the cervical dilation ⁇ 3 cm. The onset of active phase was defined as cervical dilation ⁇ 3cm.
  • the 1 st (pre-labor) and 2 nd (latent-phase) blood samples were collected at a median (interquartile, IQR) time of 13.6 h (10.9 h -22.9 h) and 7.08 h (4.99 h -11.7 h) before delivery, respectively.
  • the 1 st and 2 nd blood samples were collected at a median (IQR) time of 18.2 h (9.82 h –25.6 h) and 7.37 h (4.37 h –11.4 h) before delivery, respectively.
  • RNA-seq study to identify delivery-associated circulating transcripts
  • transcripts Compared with the pre-labor blood samples, 3, 620 differentially expressed (428 decreased and 3, 192 increased) transcripts were identified in the latent-phase samples (Wilcoxon signed rank test, False Discovery Rate (FDR) -adjusted p-values ⁇ 0.05 and
  • DACTs delivery-associated circulating transcripts
  • RT-qPCR assays targeting five down-and five up-regulated transcripts with the smallest Wilcoxon adjusted p-values were ordered (Integrated DNA Technologies, Inc. ) for the validation phase. Further, reference genes with stable RNA levels in blood were shortlisted from the transcript-and exon-level data (Table 6) . EYA3 and CNOT1 were chosen based on a reasonably small biological coefficient of variation and an RNA level that is similar in magnitude compared to the ten transcripts selected above for RT-qPCR analysis.
  • IQR The median (IQR) log 2 normalized RNA levels of the ten target RNA transcripts in maternal blood before labor and during latent phase were shown in Table 7 and Fig. 1B.
  • IQR The median log 2 normalized RNA levels of the ten target RNA transcripts in maternal blood before labor and during latent phase were shown in Table 7 and Fig. 1B.
  • the five transcripts with decreased levels during labor detected by RNA-seq namely GMEB2, PDCD11, IGLV1-36, UPF2 and TRBV15 mRNA
  • their log 2 fold change measured by RT-qPCR ranged from -0.50 to -4.36 (Fig. 1B, first row of panels) .
  • RNA-seq a subset of the five transcripts with increased levels detected by RNA-seq, namely TRAPPC11, DHRS13, SH3BP5, CA4 and PADI4 mRNA
  • the maternal blood levels of these tested transcripts identified in the RNA-seq study were validated to be differentially expressed during labor also in the RT-qPCR study.
  • lower levels of a down-regulated transcript is related to a shorter time interval from blood sampling to onset or delivery.
  • No significant correlation was observed for the time between collection of the 1 st sample and onset or delivery for other down-regulated transcripts or any up-regulated transcripts in the RT-qPCR study.
  • no correlation was observed for the time between collection of the 2 nd sample and onset or delivery (Figs. 1F and 1G) .
  • RoC [ (log 2 latent-phase RNA levels) - (log 2 pre-labor RNA levels) ] /time between 1 st and 2 nd samples. Then, this RoC value and the time between blood sampling and onset or delivery were tested for correlation.
  • RoFC [ (log 2 latent-phase RNA levels) / (log 2 pre-labor RNA levels) ] /time between 1 st and 2 nd samples.
  • Tables 11A-D (7-8, 11-12) , which summarize the Kaplan-Meier survival analysis of RoFC data.
  • RNA level was used to categorize the women as having a low or high level/RoC of the concerned transcript (Tables 9, 4, 10, 5, 11) . This threshold was the RNA level (e.g., Table 9, column 5) of a given percentile (column 6) of all women in the concerned study.
  • the PDCD11 transcript assay could be used to predict delivery within 10 hours after blood sampling (100-fold, mean sensitivity 0.870 (95%CI, 0.800 –0.940) , mean specificity 0.795 (95%CI, 0.760 –0.830) , mean accuracy 0.810 (95%CI, 0.780 –0.840) (Fig. 4B) .
  • the whole differentially expressed transcripts were associated with 192 biological processes (176 over-represented and 16 under-represented) .
  • Figure 5A shows the over-/under-represented processes with 10 lowest FDR or 10 highest fold enrichment scores.
  • 3192 of the 3620 differentially expressed transcripts were identified to be upregulated during the latent phase of labor, which were associated with 188 biological processes (175 over-represented and 13 under-represented) .
  • Figure 5B shows the over-/under-represented biological processes with either 10 lowest FDR or 10 highest fold enrichment score.
  • the remaining 428 transcripts were identified to be downregulated during the latent phase of labor, which were associated with 11 biological processes (10 over-represented and 1 under-represented) ( Figure 5C) .
  • FIG. 5D shows the over-represented processes with 10 lowest FDR and 10 highest fold enrichment and all the under-represented processes. 1308 of the 1497 differentially expressed transcripts were identified to be upregulated during the latent phase of labor, which were associated with 39 biological processes (33 over-represented and 6 under-represented) .
  • Figure 5E shows the over-represented processes with either 10 lowest FDR or 10 highest fold enrichment and all the under-represented processes. The remaining 189 transcripts were identified to be downregulated, and no significant biological processes were found in the GO enrichment analysis.
  • RNA transcription For the five selected genes that showed decreased RNA transcript levels as measured by RT-qPCR, in maternal blood during labor, three of them are related to the key aspects of RNA regulation: RNA transcription, mRNA nuclear export and rRNA maturation.
  • GMEB2 gene is a member of the KDWK gene family of combinatorial transcription modulators (18) . It also has a role in modulating the properties of glucocorticoid receptor-mediated transactivation (19-22) . In a study comparing the distribution of GMEB2 cDNA across 24 different human tissues using PCR technique, expression of GMEB2 was found to be high in placenta (23) .
  • PDCD11 protein is involved in a major pathway of rRNA processing in the nucleus and cytosol, essential for the generation of mature 18S rRNA (24, 25) .
  • UPF2 protein is a part of a post-splicing multiprotein complex involved in both mRNA nuclear export and a process called nonsense-mediated mRNA decay (NMD) (26-28) . It is a eukaryotic mRNA surveillance mechanism, which recognizes premature termination codons in transcripts and initiates degradation of these transcripts.
  • IGLV1-36 encodes the V region of the variable domain of immunoglobulin light chains, which is a part of the antigen binding site (29) .
  • the variable domains of the immunoglobulin is subjected to somatic hypermutations after exposure to antigens and thus participates in the antigen recognition process.
  • TRBV15 is predicted to be a part of the human beta T cell receptor (TCR) , and thus involved in the cell surface receptor signaling pathway (30) .
  • TRAPPC11 is involved in endoplasmic reticulum to Golgi vesicle-mediated transport, and constitutive secretory pathway (31, 32) .
  • Subjects with variants of TRAPPC11 displayed membrane trafficking defects (33) .
  • DHRS13 belongs to the large short-chain dehydrogenase/reductase (SDR) family of enzymes that involve in the metabolism of steroid hormones, prostaglandins, retinoids, lipids, and xenobiotics. DHRS13 is located in membrane, and it shares highest similarity with retinol dehydrogenase. It is predicted to enable NADP-retinol dehydrogenase activity and involved in retinal metabolic process (34) . It is also found in the membrane of NK cells (35) .
  • SDR small short-chain dehydrogenase/reductase
  • SH3BP5 is related to pathways in immune response Fc epsilon RI pathway. It interacts with Bruton’s tyrosine kinase (BTK) , inhibiting the auro-and transphosphorylation activity of BTK (36) and plays a negative regulatory role in BTK-related cytoplasmic signaling in B-cells (37) . It is also suggested to be involved in B-cell receptor induced apoptotic cell death (37) . SH3BP5 also interacts with several mitogen-activated protein kinase (MAPK) (38) . It is highly expressed in reproductive organs (testis and ovary) (36) .
  • MAPK mitogen-activated protein kinase
  • CA4 belongs to a large family of carbonic anhydrases that catalyze the reversible hydration of carbon dioxide. They are involved in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid (39) .
  • CA4 is found in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules. It is suggested to be transported via the classical biosynthesis or secretory pathway (40) .
  • PADI protein plays a key role in regulating arginine methylation and citrullination in histones (41-44) .
  • NETs neutrophil extracellular traps
  • TRAPPC11 is related to ER to Golgi vesicle-mediated transport
  • DHRS13 is also located in the membrane of NK cells
  • CA4 is located in ER-Golgi intermediate compartment.
  • the exclusion criteria included women complicated by multi-fetal gestation, macrosomia (estimated fetal weight ⁇ 4200g) (47) or other contraindications for vaginal delivery; fetuses with chromosomal or structural malformation; the presence of infection or significant medical conditions such as systematic lupus erythematosus; or recent use of antibiotics or vaccination; and cesarean section.
  • RNA preservative RNA-later, Thermo Fisher Scientific
  • the sequencing reads were mapped and quantified at transcript and exon levels. Count data with 205870 transcripts were generated. Normalized RNA levels expressed in transcript per million (TPM) was applied.
  • Paired t-test was performed on all transcript counts. Changes were detected in 9471 transcripts exons between before onset and latent phase samples. Transcripts with paired t-test p-value > 0.05 were removed before performing adjustment for multiple testing comparison. Wilcoxon signed rank test and adjustment by FDR method were then performed, and 3970 transcripts were differentially expressed.
  • transcripts with median levels changed by > 1.1-fold were selected, and 3620 of them fulfilled the criteria.
  • the transcript and exon data were then integrated and markers that were significant in adjusted p-values and concordant in direction of change at both transcript and exon-level were retained. Only 266 markers were selected. Thirty up-regulated markers with the smallest adjusted p-values were further selected and all 10 down-regulated markers were included.
  • Exon markers with normalized count below 20 and coefficient of variations below 50 were selected and their stability values were calculated using NormFinder in R (v5) . In NormFinder, the lower the stability values indicate the more stable gene.
  • RT-qPCR Reverse-transcriptase quantitative PCR
  • RT-qPCR assays were designed, optimized and performed in compliance with the MIQE guidelines (48) .
  • dual-labeled hydrolysis probes (6-FAM at 5’ end and Iowa Blackhole Quencher at 3’ end) and primers were designed to be intron-spanning whenever possible to avoid detecting any residual genomic DNA.
  • Primers and probes were synthesized by Integrated DNA Technologies (IDT) . Sequences and information of the primers and probes are listed in Table 12.
  • RT-qPCR experiments were performed on LightCycler LC480 (Roche) with TaqMan TM Universal Master Mix II, with UNG (ThermoFisher Scientific) . No template controls were included in parallel for each run. Details of the experiment including thermal profile are described in Table 12.
  • Absolute quantitative methods were used to quantify the concentrations of RNA transcripts using a calibration curve of serially-diluted standards. To control for varying RNA input, we have normalized the concentration of the test RNA transcript to that of a list of reference genes (Table 6) , which has stable concentrations across different physiological conditions.
  • RNA-seq analysis To test for differences in maternal blood levels of targeted RNA transcripts before and after onset of labor as measured by RT-qPCR, the Wilcoxon signed-rank test was used. A target transcript is considered validated if significant difference was detected in samples before and after onset of labor, with the direction of change was concordant with that in the RNA-seq analysis.
  • Kaplan-Meier survival analysis was performed, using (i) time between blood-sampling and onset of active phase; and (ii) time between blood-sampling and delivery.
  • a threshold was determined to separate the cohort into two groups: (i) women with blood levels of RNA transcript before onset of labor greater than or equal to the threshold, and (ii) women with blood levels of RNA transcript before onset of labor less than the threshold.
  • Log-rank test was performed to compare the Kaplan-Meier curves between two groups for each transcript.
  • a transcriptome-wide gene set enrichment analysis was performed to cluster gene ontology (GO) annotated biological process using the online Panther software (version 16.0) with FDR correction (49) .
  • Gene ontology annotation specifically categorizes the genes’ associated biological processes by comparing them to the whole human genome list.
  • Several iterations of this analysis were performed including the whole list of differentially expressed transcripts the present inventors identified using RNA sequencing and sub-lists based on the direction of change and the fold changes.
  • Significant GO-terms (FDR ⁇ 0.05) associated with this gene list were presented with the fold enrichment and FDR.
  • Fold enrichment is calculated by comparing the frequency of our genes that were annotated to certain terms to the background frequency representing the number of genes in the human genome that falls under the same terms. Fold enrichment greater than 1 represented a greater frequency, whereas fold enrichment less than 1 represented a lower frequency, in this list compared to the human genome set.
  • JNK c-Jun N-terminal kinase
  • Table 1 a including intrauterine growth restriction, hypertension, diabetes, and oligohydramnios b: including precipitate labor, postpartum haemorrhage *: Mann-Whitney rank sum test for continuous variables, and Chi-square test for categorical variables
  • Threshold of maternal blood transcript level or rate of change shown as percentile value of the RT-qPCR study population
  • Threshold of transcript level or rate of change (shown as normalized value of the RT-qPCR study population)
  • Table 11 The use of delivery-associated circulating transcripts for the prediction of onset of labor and delivery. Women with level or rate of change of transcript specified by the threshold (A, B) as high or low showed different time-to-event in parturition (C and D, respectively) .
  • Table 12A Oligonucleotide sequences of forward and reverse PCR primers and hydrolysis probes for RT-qPCR assays for quantification of delivery-associated circulating transcripts (DACTs) and reference genes. All sequences for forward (-F) and reverse (-R) PCR primers and hydrolysis probes (-P) are listed from the 5' end to the 3' end. For probe, the 5' end was labeled with 6-carboxyfluorescein (/56-FAM/) and 3' end with Iowa Blackhole Quencher FQ (/3IABkFQ/) and internally with the ZEN internal quencher (/ZEN/) (Integrated DNA Technologies, Coralville, IA USA) .
  • Table 12B Reaction conditions for RT-qPCR assays for quantification of delivery-associated circulating transcripts (DACTs) and reference genes.
  • Thermocycler-detector 480 System (Roche)
  • Reaction conditions of qPCR assays Volumes are in ⁇ L with total volume for each reaction made up to 10 ⁇ L using RNase-free water.
  • Table 12C Thermal profile for RT-qPCR assays for quantification of delivery-associated circulating transcripts (DACTs) and reference genes.

Landscapes

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

Abstract

La présente invention concerne des procédés utiles pour prédire le moment du début du travail et de la naissance de l'enfant chez les femmes enceintes, fondés sur l'analyse du niveau de l'un des biomarqueurs d'ARN nouvellement identifiés circulant dans le sang de la femme. La présente invention concerne également des compositions, des kits et des dispositifs utiles pour les procédés d'évaluation du moment du début du travail et de la naissance de l'enfant.
PCT/CN2024/115026 2023-09-12 2024-08-28 Marqueurs circulants pour la parturition Pending WO2025055728A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363538022P 2023-09-12 2023-09-12
US63/538,022 2023-09-12

Publications (1)

Publication Number Publication Date
WO2025055728A1 true WO2025055728A1 (fr) 2025-03-20

Family

ID=95020928

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/115026 Pending WO2025055728A1 (fr) 2023-09-12 2024-08-28 Marqueurs circulants pour la parturition

Country Status (1)

Country Link
WO (1) WO2025055728A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005059557A1 (fr) * 2003-12-16 2005-06-30 Monash University Procedes de prediction, de diagnostic et de surveillance d'un accouchement a terme ou avant terme
CN1723289A (zh) * 2003-01-17 2006-01-18 香港中文大学 作为妊娠相关病症的诊断标志物的循环mRNA
CN106232833A (zh) * 2014-01-30 2016-12-14 加利福尼亚大学董事会 用于非侵入性诊断的甲基化单体型分析(monod)
RU2682713C1 (ru) * 2018-03-13 2019-03-21 Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр акушерства, гинекологии и перинатологии имени академика В.И. Кулакова" Министерства здравоохранения Российской Федерации Способ прогнозирования преждевременных родов путем совместного определения внеклеточной ДНК и интерлейкина-8 в плазме периферической крови
CN109890984A (zh) * 2016-10-19 2019-06-14 香港中文大学 通过母体血浆dna的甲基化和大小谱分析进行胎龄评估
CN111566228A (zh) * 2017-10-23 2020-08-21 陈扎克伯格生物中心公司 用于在胎儿孕育中预测胎龄和早产的无创分子钟
WO2023081768A1 (fr) * 2021-11-04 2023-05-11 Mirvie, Inc. Procédés et systèmes pour déterminer un état associé à la grossesse d'un sujet
US20230265522A1 (en) * 2020-04-20 2023-08-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. Multi-gene expression assay for prostate carcinoma

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1723289A (zh) * 2003-01-17 2006-01-18 香港中文大学 作为妊娠相关病症的诊断标志物的循环mRNA
WO2005059557A1 (fr) * 2003-12-16 2005-06-30 Monash University Procedes de prediction, de diagnostic et de surveillance d'un accouchement a terme ou avant terme
CN106232833A (zh) * 2014-01-30 2016-12-14 加利福尼亚大学董事会 用于非侵入性诊断的甲基化单体型分析(monod)
CN109890984A (zh) * 2016-10-19 2019-06-14 香港中文大学 通过母体血浆dna的甲基化和大小谱分析进行胎龄评估
CN111566228A (zh) * 2017-10-23 2020-08-21 陈扎克伯格生物中心公司 用于在胎儿孕育中预测胎龄和早产的无创分子钟
RU2682713C1 (ru) * 2018-03-13 2019-03-21 Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр акушерства, гинекологии и перинатологии имени академика В.И. Кулакова" Министерства здравоохранения Российской Федерации Способ прогнозирования преждевременных родов путем совместного определения внеклеточной ДНК и интерлейкина-8 в плазме периферической крови
US20230265522A1 (en) * 2020-04-20 2023-08-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. Multi-gene expression assay for prostate carcinoma
WO2023081768A1 (fr) * 2021-11-04 2023-05-11 Mirvie, Inc. Procédés et systèmes pour déterminer un état associé à la grossesse d'un sujet

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GOMEZ-LOPEZ NARDHY, ROMERO ROBERTO, GALAZ JOSE, BHATTI GAURAV, DONE BOGDAN, MILLER DEREK, GHITA CORINA, MOTOMURA KENICHIRO, FARIAS: "Transcriptome changes in maternal peripheral blood during term parturition mimic perturbations preceding spontaneous preterm birth", BIOLOGY OF REPRODUCTION, NEW YORK, NY [U.A.] : ACADEM. PRESS, US, vol. 106, no. 1, 13 January 2022 (2022-01-13), US , pages 185 - 199, XP093292816, ISSN: 0006-3363, DOI: 10.1093/biolre/ioab197 *

Similar Documents

Publication Publication Date Title
US20240038396A1 (en) Methods and systems for determining risk of a pregnancy complication occurring
Hromadnikova et al. Absolute and relative quantification of placenta-specific microRNAs in maternal circulation with placental insufficiency–related complications
JP5800471B2 (ja) 出生前診断およびモニタリングのためのマーカー
Farina et al. High levels of fetal cell-free DNA in maternal serum: a risk factor for spontaneous preterm delivery
EP3701043B1 (fr) Horloge moléculaire non invasive relative au développement foetal et prédisant l'âge gestationnel et l'accouchement prématuré
US10947593B2 (en) Biomarkers for premature birth
Whitehead et al. Identifying late-onset fetal growth restriction by measuring circulating placental RNA in the maternal blood at 28 weeks’ gestation
Whitehead et al. Quantifying circulating hypoxia-induced RNA transcripts in maternal blood to determine in utero fetal hypoxic status
Hromadnikova Extracellular nucleic acids in maternal circulation as potential biomarkers for placental insufficiency
Adel et al. Evaluation of MicroRNA-210 and Protein tyrosine phosphatase, non-receptor type 2 in Pre-eclampsia
Rokni et al. Association between miRNA-152 polymorphism and risk of preeclampsia susceptibility
Dong et al. Expression of lncRNA MIR193BHG in serum of preeclampsia patients and its clinical significance
US10954564B2 (en) Combinations of cell free nucleic acids
US11149315B2 (en) Method for predicting cervical shortening and preterm birth
Turner et al. Detection of fetal Rhesus D gene in whole blood of women booking for routine antenatal care
US20250270647A1 (en) Combinations of cell free nucleic acids
WO2025055728A1 (fr) Marqueurs circulants pour la parturition
Guo et al. The effect of MicroRNAs variants on idiopathic recurrent pregnancy loss
EP3551771B1 (fr) Procédé pour la prédiction du risque de thrombose veineuse profonde et d'embolie pulmonaire associée à des préparations hormonales et à des niveaux hormonaux
CN107447008B (zh) 用于诊断不明原因复发性流产的增强子rna组合、引物组及应用和试剂盒
WO2025011401A1 (fr) Marqueurs d'arn circulant à usage diagnostique
Luque Gómez et al. Usefulness of circulating microRNAs for the prediction of early preeclampsia at first-trimester of pregnancy

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: 24864450

Country of ref document: EP

Kind code of ref document: A1