阅读说明：本技术 早产的生物标记 (Biomarkers for preterm labor ) 是由 N·阿贝尔 廖善敏 林向前 E·鲁宾 于 2018-10-30 设计创作，主要内容包括：本发明涉及生物标记,特别地,尽管并非唯一,涉及检测早产的生物标记。该生物标记适于,在出生前数周或数月,区分个体在妊娠37周前的生育风险。(The present invention relates to biomarkers and in particular, although not exclusively, to detecting biomarkers of preterm labor. The biomarker is suitable for distinguishing individuals at risk of birth by week 37 of gestation, weeks or months prior to birth.)

1. A method for predicting whether an individual is at risk of preterm birth, the method comprising determining the level of a biomarker in a sample taken from the individual, and predicting whether the individual is at risk of preterm birth based on the level of the biomarker, wherein the biomarker is selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2.

2. The method of claim 1, wherein the sample is a vaginal fluid sample.

3. The method of claim 1 or 2, wherein the biomarker is a protein.

4. The method of any one of claims 1 to 3, wherein the level of the biomarker is compared to a reference level, wherein the reference level is derived from the level of the biomarker in a sample taken from an individual known to have undergone preterm or term delivery.

5. The method of any one of claims 1 to 4, further comprising predicting the risk of preterm labor with one or more other preterm birth indicators selected from the group consisting of fetal fibronectin testing, contractions, vaginal bleeding, fluid exudation from the vagina, increased vaginal discharge, back pain, and lower abdominal cramps.

6. Progesterone for use in the treatment of an individual expected to be at risk of preterm birth, wherein the individual has been predicted to be at risk of preterm birth using a method according to any one of claims 1 to 4.

7. A method of selecting a treatment for an individual to reduce the risk of preterm birth, the method comprising predicting the risk of preterm birth for an individual using the method of any one of claims 1-4, and administering a treatment to reduce the risk of preterm birth if said individual is determined to be at risk of preterm birth, wherein the treatment for reducing the risk of preterm birth comprises progesterone and/or cervical cerclage.

8. A method for predicting whether an individual is at risk of preterm birth, the method comprising determining the level of a biomarker in a sample taken from the individual, and communicating the determined amount to a physician involved in the treatment of the individual, wherein the risk of preterm birth is predicted based on the level of the biomarker in the sample, and wherein the biomarker is selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2.

9. The method of any one of claims 1 to 8, wherein the method for predicting whether the individual is at risk of preterm birth is a computer-implemented method.

10. A method for detecting ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2, the method comprising:

a. obtaining a vaginal fluid sample from an individual;

b. detecting whether ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is present in the vaginal fluid sample by contacting the vaginal fluid sample with an anti-ECM 1 antibody, an anti-FGA antibody, an anti-EFEMP 1 antibody, an anti-GGH antibody, an anti-PEDF antibody, an anti-PTN antibody, or an anti-LAMC 2 antibody and detecting binding between ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 and the antibody.

11. A method for determining the risk of preterm birth in an individual, the method comprising:

a. obtaining a sample from an individual;

b. detecting whether ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is present in the sample by contacting the sample with an anti-ECM 1 antibody, an anti-FGA antibody, an anti-EFEMP 1 antibody, an anti-GGH antibody, an anti-PEDF antibody, an anti-PTN antibody, or an anti-LAMC 2 antibody and detecting binding between ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 and the antibody; and

c. determining that the individual is at risk of preterm birth when the presence of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is detected in the sample.

12. The method of claim 11, wherein the sample is a vaginal fluid sample.

13. A method of determining the risk of preterm birth in an individual and prolonging pregnancy in an individual, the method comprising:

a. obtaining a sample from an individual;

b. detecting whether ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is present in the vaginal fluid sample;

c. determining that the individual is at risk of preterm birth when the presence of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is detected in the vaginal fluid sample; and

d. administering an effective amount of progesterone to an individual determined to be at risk of preterm birth, or if an individual is determined to be at risk of preterm birth, selecting the individual for treatment with an effective amount of one or more agents selected from progesterone or analogs thereof, tocolytics, corticosteroids, antibiotics, NSAIDs, or omega 3 fatty acids or derivatives thereof; and/or

e. The cervical cerclage is performed on an individual identified as at risk for preterm birth, or if the individual is identified as at risk for preterm birth, the individual is selected for cervical cerclage.

14. The method of any one of claims 10 to 13, wherein the level of the biomarker is determined using an ELISA.

15.A kit for predicting the risk or likelihood of preterm birth in a subject, the kit comprising an anti-ECM 1 antibody, an anti-FGA antibody, an anti-EFEMP 1 antibody, an anti-GGH antibody, an anti-PEDF antibody, an anti-PTN antibody, or an anti-LAMC 2 antibody.

Technical Field

The present invention relates to biomarkers and in particular, although not exclusively, to detecting biomarkers of preterm labor. The biomarker is suitable for distinguishing individuals at risk of birth by week 37 of gestation, weeks or months prior to birth.

Background

Preterm birth is defined as birth prior to 37 weeks of gestation. It is estimated that more than 1500 million infants are born prematurely each year. Preterm birth is one of the leading causes of death in children under five years of age worldwide, with an estimated million cases of death associated with preterm birth. Many survivors face the challenge of lifelong disability, including learning disabilities and visual and auditory problems. Although advances in neonatal science have improved the survival rate of premature labor over the past decades, more than 20% of premature newborns suffer from at least one serious disability, including chronic lung disease, impaired mental development, cerebral palsy (cerebral palsy), deafness, or blindness.

There is an urgent need to identify pregnant women at risk of preterm birth. In the present example, treatment of high risk pregnancies involves prophylactic treatment or intensive or close monitoring of pregnancies to reduce early yields. However, in most cases, the high risk classification of pregnancy is due to past medical history or clinical examination, and only a small fraction of true high risk pregnancies can be determined to be prone to preterm birth. Most preterm pregnancies are still unrecognizable at an early stage, and thus early medical intervention in these cases is not possible.

There are several preterm delivery risk assessment tests on the market for women with risk symptoms. One example is the Fetal Fibronectin (fFN) test, which provides a risk assessment for symptomatic women. If preterm birth is likely to occur, fetal fibronectin will be present in the vagina; therefore, the fn test is commonly used in pregnant women who develop possible symptoms of premature birth, such as contractions, vaginal bleeding, fluid exudation from the vagina, increased vaginal discharge, back pain, and lower abdominal cramps. The strength of the fn test is a high negative predictive value for up to 10 days after testing (i.e., a negative result means that the likelihood of preterm birth within 7 to 10 days after testing is low). However, when the fn test is positive, the results are less determinable.

Patient management varies with risk factors. Prophylactic treatments such as progesterone have been shown to reduce early productivity in a number of clinical studies that rank women with short cervical lengths or a history of preterm birth as high risk groups. Symptomatic women may receive either tocolytic or steroid treatment based on risk factors. The limitation of clinical practice today is that the relevance of treatment to prognosis is very low.

Therefore, there is an urgent need to efficiently identify pregnancies with a high risk of preterm birth in order to administer appropriate treatment in a timely manner to reduce early productivity. The present invention provides a biomarker and method for predicting the risk of preterm birth, to at least partially overcome some of the disadvantages. In particular, the present invention seeks to provide a risk assessment for classifying women at high risk of preterm birth within weeks or even months prior to the onset of preterm symptoms.

The present invention was devised in view of the above considerations.

Summary of The Invention

The present invention provides novel biomarkers and methods for predicting the risk or likelihood of preterm birth in a subject. The biomarkers disclosed herein are identified by metadata analysis (meta-data analysis) and subsequently validated in samples derived from patients. The biomarkers disclosed herein differentiate term production from samples from preterm individuals weeks or months prior to the onset of symptoms in the individual. Such biomarkers can be used to identify the risk of preterm birth in an individual and thus can be used to guide clinical decisions such as initiating treatment to extend pregnancy and/or prevent or reduce the risk of preterm birth.

The methods disclosed herein are useful for determining the risk or likelihood of preterm birth in asymptomatic or symptomatic individuals. In particular embodiments, the subject is asymptomatic.

ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2 are biomarkers of preterm labor as disclosed herein. Each of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2 may be used in a method for identifying an individual at risk of preterm birth, and for determining whether an individual is at risk of preterm birth or for predicting whether an individual is at risk of preterm birth. A change in the level of the biomarker compared to a control or reference level may be indicative that the individual is at risk of preterm birth. The methods involve determining the amount of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2 in a sample from a test individual. In some aspects, the methods involve determining the amount of all biomarkers ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2, and predicting the risk of preterm birth.

Low expression of specific biomarkers including ECM1, GGH, LAMC2, EFEMP1, PTN, and FGA may indicate that the individual is at risk for preterm birth.

The individual may be at risk of preterm birth when certain biomarkers are overexpressed, including GGH, LAMC2, EFEMP1, PTN, FGA, and PEDF.

In some cases, if a biomarker is overexpressed at a particular point in pregnancy, or is underexpressed at a different point in pregnancy, it may be indicative that the individual is at risk of preterm birth.

The present invention provides a method of predicting whether an individual is at risk for preterm birth, the method comprising determining the level of a biomarker in a sample taken from the individual, and classifying the individual as at risk for preterm birth or not based on the biomarker value, wherein the biomarker is selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. The method may further comprise administering treatment to an individual determined to be at risk. The treatment may comprise cervical cerclage or administration of one or more agents selected from progesterone or an analog thereof, a tocolytic agent (tocolytic), a corticosteroid, an antibiotic, an NSAID, or an omega 3 fatty acid or derivative thereof. The progesterone can be a synthetic progesterone, such as 17-alpha-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

Also disclosed is a method of determining the likelihood that an individual will experience preterm birth, comprising measuring a biomarker value selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2 in a sample from the individual, and determining the percent likelihood that an individual will experience preterm birth based on the biomarker value.

A computer-implemented method for predicting whether an individual is at risk of preterm birth, the method comprising retrieving biomarker information for the individual on a computer. The information comprises biomarker values corresponding to at least one biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2, and the biomarker values are classified by the computer; based on the classification, a likelihood that the individual is at risk of preterm birth is indicated.

Also disclosed is a method of treatment comprising administering one or more agents selected from the group consisting of progesterone or analog thereof, an anti-miscarriage agent, a corticosteroid, an antibiotic, an NSAID, or an omega 3 fatty acid or derivative thereof, to an individual identified as at risk of preterm birth based on a value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. The invention also discloses the progesterone used in the method and application of the progesterone in preparing a medicament used in the method. The progesterone can be a synthetic progesterone, such as 17-alpha-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

Also disclosed is a method of treatment comprising determining cervical cerclage in an individual at risk of preterm birth based on the value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. Methods for selecting an individual for cervical cerclage therapy are also disclosed.

Also disclosed is an anti-miscarriage agent or a steroid for use in a method of treatment of an individual predicted to be at risk of preterm birth, wherein the individual is determined to be at risk of preterm birth based on the value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. Also disclosed is a method of treatment comprising administering an anti-miscarriage agent or a steroid to an individual identified as at risk for preterm birth, wherein the individual is identified as at risk for preterm birth based on a value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2.

In this regard, the present invention provides the use of an anti-miscarriage agent or a steroid for the manufacture of a medicament for the treatment of an individual determined to be at risk of preterm birth, wherein the individual is predicted to be at risk of preterm birth based on a value for a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2.

The tocolytic agent or steroid is used, or the medicament may be used in a method involving determining a biomarker value selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2, and determining the risk of preterm birth based on the biomarker value.

Also disclosed are methods of treatment of an individual at risk of preterm birth with one or more agents selected from the group consisting of progesterone or an analog thereof, an anti-miscarriage agent, a corticosteroid, an antibiotic, an NSAID, or an omega 3 fatty acid or derivative thereof, for predicting risk of preterm birth, wherein the individual is determined to be at risk of preterm birth based on the value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. The progesterone can be a synthetic progesterone, such as 17-alpha-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

Also disclosed is a method of treatment comprising administering one or more agents selected from the group consisting of progesterone or analog thereof, an anti-abortion agent, a corticosteroid, an antibiotic, an NSAID, or an omega 3 fatty acid or derivative thereof, to an individual identified as at risk of preterm birth, wherein said individual is identified as at risk of preterm birth based on a value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. The progesterone can be a synthetic progesterone, such as 17-alpha-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

The invention also provides the use of one or more agents selected from the group consisting of progesterone or analog thereof, an anti-miscarriage agent, a corticosteroid, an antibiotic, an NSAID, or an omega 3 fatty acid or derivative thereof, for the manufacture of a medicament for the treatment of an individual determined to be at risk of preterm birth, wherein the individual is determined to be at risk of preterm birth based on a value of a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. The progesterone can be a synthetic progesterone, such as 17-alpha-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

The agent or medicament is for use or is useful in a method involving determining a value for a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2, based on which the risk of preterm birth can be determined. The invention provides a method for detecting ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2, the method comprising:

a. obtaining a vaginal fluid sample from an individual;

b. detecting the presence of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 in a vaginal fluid sample by contacting the vaginal fluid sample with an anti-ECM 1 antibody, an anti-FGA antibody, an anti-EFEMP 1 antibody, an anti-GGH antibody, an anti-PEDF antibody, an anti-PTN antibody, or an anti-LAMC 2 antibody and detecting the binding between ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 and the antibody.

There is also provided a method for determining the risk of preterm birth in an individual, the method comprising:

a. obtaining a sample from an individual;

b. detecting whether ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is present in the sample by contacting the sample with an anti-ECM 1 antibody, an anti-FGA antibody, an anti-EFEMP 1 antibody, an anti-GGH antibody, an anti-PEDF antibody, an anti-PTN antibody, or an anti-LAMC 2 antibody and detecting the binding between ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 and the antibody; and

c. determining that the individual is at risk of preterm birth when ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is detected in the vaginal fluid sample.

In some cases, the antibody is derived from a mouse, rabbit, or goat, preferably a mouse or rabbit. The antibody may be human, humanized, or chimeric. Preferably, the sample is a vaginal fluid sample. The vaginal fluid sample may be a cervicovaginal fluid sample. Alternatively, the sample is an amniotic fluid sample.

A method of determining the risk of preterm birth in an individual and prolonging pregnancy in an individual, the method comprising:

a. obtaining a sample from an individual;

b. detecting whether ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is present in the plasma sample;

c. determining that the individual is at risk of preterm birth when the presence of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 is detected in the vaginal fluid sample; and

d. administering to an individual determined to be at risk of preterm birth an effective amount of one or more agents selected from the group consisting of progesterone or an analog thereof, an anti-miscarriage agent, a corticosteroid, an antibiotic, an NSAID, or an omega 3 fatty acid or derivative thereof, or if the individual is determined to be at risk of preterm birth, selecting the individual and treating with an effective amount of an anti-miscarriage agent or a steroid. The progesterone can be a synthetic progesterone, such as 17-a-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

In one aspect, the invention discloses a kit for predicting the risk or likelihood of preterm birth in a subject, the kit comprising an anti-ECM 1 antibody, an anti-FGA antibody, an anti-EFEMP 1 antibody, an anti-GGH antibody, an anti-PEDF antibody, an anti-PTN antibody, or an anti-LAMC 2 antibody. Preferably, the anti-ECM 1 antibody, anti-FGA antibody, anti-EFEMP 1 antibody, anti-GGH antibody, anti-PEDF antibody, anti-PTN antibody, or anti-LAMC 2 antibody is capable of selectively binding to ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC 2.

Certain aspects of the present disclosure describe computer-implemented methods of determining the risk of preterm birth in an individual. The method may involve providing a corresponding amount of data for at least one of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, or LAMC2 in a sample taken from an individual; by computer, classification of biomarker values; and determining the risk of preterm birth for the individual based on the classification.

The present invention includes combinations of the various aspects and preferred features described unless such combinations are clearly not allowed or explicitly avoided.

Brief Description of Drawings

Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings. Wherein:

figure 1 this graph represents the difference in the amount of ECM1 between term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay with the adjusted total protein concentration. Full term (n-136) and preterm (n-64) samples were collected between 19-37 weeks of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

Figure 2 this graph represents the difference in the amount of ECM1 between term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, the number of gestation weeks 18+0-23+6 days (18-24), the number of gestation weeks 24+0-27+6 days (24-28), the number of gestation weeks 28+0-31+6 days (28-32), and the number of gestation weeks 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 3 this graph represents the difference in the amount of ECM1 between term and preterm delivery in women, as measured by time from sample collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The mean value of each group was calculated.

FIG. 4 is a graph representing the difference in GGH levels between term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay with the adjusted total protein concentration. Full term (n-136) and preterm (n-64) samples were collected between 19-37 weeks of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

FIG. 5 is a graph showing the difference in GGH levels between term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, the number of gestation weeks 18+0-23+6 days (18-24), the number of gestation weeks 24+0-27+6 days (24-28), the number of gestation weeks 28+0-31+6 days (28-32), and the number of gestation weeks 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 6 is a graph showing the difference in GGH levels between term and preterm delivery in women, depending on the time from sample collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The mean value of each group was calculated.

FIG. 7 this graph represents the difference in LAMC2 levels between full term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay with the adjusted total protein concentration. Full term (n-136) and preterm (n-64) samples were collected between 19-37 weeks of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

Figure 8 this graph represents the difference in the amount of LAMC2 between term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, week number of pregnancy 18+0-23+6 days (18-24), week number of pregnancy 24+0-27+6 days (24-28), week number of pregnancy 28+0-31+6 days (28-32), and week number of pregnancy 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 9 is a graph showing the difference in LAMC2 levels between full term and preterm labor in women, based on time from sample collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The mean value of each group was calculated.

Figure 10 this graph represents the difference in the amount of EFEMP1 between term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay adjusted for total protein. Full term (n-136) and preterm (n-64) samples were collected between 19-37 weeks of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

Figure 11 this graph represents the difference in the amount of EFEMP1 between full term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, the number of gestation weeks 18+0-23+6 days (18-24), the number of gestation weeks 24+0-27+6 days (24-28), the number of gestation weeks 28+0-31+6 days (28-32), and the number of gestation weeks 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 12 is a graph showing the difference in the amount of EFEMP1 between term and preterm delivery in women, as measured by time from specimen collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The mean value of each group was calculated.

Figure 13 this graph represents the difference in PTN amounts between full term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay with the adjusted total protein concentration. Full term (n-136) and preterm (n-64) samples were collected between weeks 19-37 of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

FIG. 14 is a graph representing the difference in PTN amounts between term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, the number of gestation weeks 18+0-23+6 days (18-24), the number of gestation weeks 24+0-27+6 days (24-28), the number of gestation weeks 28+0-31+6 days (28-32), and the number of gestation weeks 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 15 is a graph representing the difference in PTN levels between term and preterm delivery in women, as a function of time from sample collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The mean value of each group was calculated.

FIG. 16 is a graph representing the difference in FGA levels between term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay adjusted for total protein. Full term (n-136) and preterm (n-64) samples were collected between weeks 19-37 of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

FIG. 17 is a graph showing the difference in FGA levels between term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, the number of gestation weeks 18+0-23+6 days (18-24), the number of gestation weeks 24+0-27+6 days (24-28), the number of gestation weeks 28+0-31+6 days (28-32), and the number of gestation weeks 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 18 is a graph representing the difference in FGA levels between term and preterm delivery in women, as a function of time from sample collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The mean value of each group was calculated.

Figure 19 this graph represents the difference in PEDF levels between term and preterm samples. The mean biomarker protein concentration was calculated from the results of the ELISA immunoassay adjusted for total protein. Full term (n-136) and preterm (n-64) samples were collected between weeks 19-37 of gestation and presented with mean differences across groups. Data were analyzed with student t test. P-value.

Figure 20 this figure represents the difference in PEDF levels between term and preterm samples, depending on the gestational age at the time of sampling. Samples were divided into four groups based on the time of sampling, the number of gestation weeks 18+0-23+6 days (18-24), the number of gestation weeks 24+0-27+6 days (24-28), the number of gestation weeks 28+0-31+6 days (28-32), and the number of gestation weeks 32+0-37+6 days (32-37). Each group was then divided into term and preterm samples and the mean was calculated and represented as a bar graph.

FIG. 21 is a graph representing the difference in the amount of PEDF between term and preterm delivery in women, as a function of time from sample collection to delivery. The samples were divided into 4 groups, each separated by 4 weeks, to assess the difference between term and preterm samples in terms of weeks prior to birth. The time between sampling and delivery for each group was over 12 weeks (>12), 9-12,5-8, and 1-4 weeks (9-12,5-8,1-4), respectively. The average of each group was calculated.

FIG. 22 Effect of H2O2 on cell viability and proliferation. Ect1 and End1 cells were treated with 50. mu.M, 100. mu.M, 200. mu.M, and 400. mu. M H2O2 for 24 h. MTT assay was used to assess cell activity and proliferation of Ect1 and End1, and is expressed as% to untreated control (0 μ MH2O 2). Data are presented as mean ± SEM and analyzed using student's t-assay. P <0.05, n-3 compared to the individual controls.

FIG. 23 Effect of H2O2 on ECM1 expression in Ect1 and End1 cells. Quantitation of ECM1 in (a) Ect1 and (b) End1 medium after H2O2 treatment (24H) using ELISA. Fold change in ECM1 expression after H2O2 treatment was calculated as the treated and untreated cells (control) divided by the normalized amount of ECM1 (pg/mg total protein). Data are expressed as mean ± SEM, taken from at least 4 independent experiments and analyzed using student's t-assay. P <0.05 compared to control.

FIG. 24. effect of H2O2 on LAMC2 expression in Ect1 and End1 cells. LAMC2 in (a) Ect1 and (b) End1 medium after treatment with H2O2 (24H) was quantified by ELISA. Fold change in LAMC2 expression after H2O2 treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized LAMC2 amount (pg/mg total protein). Data are expressed as mean ± SEM, taken from at least 4 independent experiments and analyzed using student's t-assay. P <0.05 compared to control.

FIG. 25 Effect of H2O2 on FGA expression in Ect1 and End1 cells. FGA in (a) Ect1 and (b) End1 medium after H2O2 treatment (24H) was quantified by ELISA. Fold change in FGA expression after H2O2 treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of FGA (ng/mg total protein). Data are expressed as mean ± SEM, taken from at least 5 independent experiments and analyzed using student's t-assay. P <0.005, compared to control.

FIG. 26. effect of H2O2 on GGH expression in Ect1 and End1 cells. GGH in (a) Ect1 and (b) End1 media after treatment with H2O2 (24H) was quantified by ELISA. Fold change in GGH expression after H2O2 treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of GGH (ng/mg total protein). Data are expressed as mean ± SEM, taken from at least 5 independent experiments and analyzed using student's t-assay.

FIG. 27 effect of LPS on ECM1 expression in Ect1 and End1 cells. Quantitation of ECM1 in (a) Ect1 and (b) End1 medium after LPS treatment (24h) using ELISA. Fold change in ECM1 expression after LPS treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of ECM1 (pg/mg total protein). Data are presented as mean ± SEM and analyzed using student's t-assay, with n-5. P <0.05 compared to individual controls.

FIG. 28. effect of LPS on LAMC2 expression in Ect1 and End1 cells. Quantification of LAMC2 in (a) Ect1 and (b) End1 medium after LPS treatment (24h) using ELISA. Fold change in LAMC2 expression after LPS treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized LAMC2 amount (pg/mg total protein). Data are presented as mean ± SEM and analyzed using student's t-assay, with n-5. P <0.05, P <0.05 compared to individual controls.

FIG. 29 Effect of LPS on GGH expression in Ect1 and End1 cells. GGH in both Ect1 and End1 media after LPS treatment (24h) was quantified using ELISA. Fold change in GGH expression after LPS treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of GGH (ng/mg total protein). Data are presented as mean ± SEM and analyzed using student's t-assay, with n-5.

FIG. 30. effect of LPS on FGA expression in Ect1 and End1 cells. FGA was quantified in both Ect1 and End1 media after LPS treatment (24h) using ELISA. Fold change in FGA expression after LPS treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of FGA (ng/mg total protein). Data are presented as mean ± SEM and analyzed using student's t-assay, with n-5.

FIG. 31. effect of H2O2 on EFEMP1 expression in Ect1 cells. EFEMP1 in Ect1 medium after H2O2 treatment (24H) was quantified using ELISA. Fold change in EFEMP1 expression after H2O2 treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of EFEMP1 (ng/mg total protein). Data are expressed as mean ± SEM, taken from at least 5 independent experiments and analyzed using student's t-assay. P < 0.05% compared to control.

Figure 32 effect of H2O2 on EFEMP1 expression in End1 cells. EFEMP1 was quantified in H2O2 treated (24H) post-End 1 medium by ELISA. Fold change in EFEMP1 expression after H2O2 treatment was calculated as the amount of treated versus untreated cells (control) divided by the normalized amount of EFEMP1 (ng/mg total protein). Data are expressed as mean ± SEM, taken from at least 5 independent experiments and analyzed using student's t-assay. P <0.005, compared to control.

Detailed Description

Aspects and embodiments of the invention will now be discussed with reference to the drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

The methods and biomarkers described herein can be used to determine whether an individual is at risk of preterm birth, identify an individual as at risk of preterm birth, or predict an individual's risk of preterm birth.

The terms "predict" and "determine" are used interchangeably herein and are used to indicate or assess that an individual will develop preterm birth. The methods disclosed herein may be used to determine or predict the likelihood (or "risk") that an individual will experience preterm birth.

Preterm delivery is the delivery of a fetus by the mother before 37 weeks of gestation. Preterm birth can be subdivided into pregnancy of 35+0 to 36+6 days late, 32+0 to 34+6 days mid-term preterm birth, and 32 weeks early preterm pregnancy.

The cause of premature birth is often unknown. The risk factors include diabetes, hypertension, more than one infant, obesity or lack of weight, various vaginal infections, smoking, psychological stress, etc.

Preeclampsia (preeclampsia) is clinically manifested by hypertension and proteinuria, manifested by 20 weeks before delivery and 6 weeks after delivery. Although preeclampsia can lead to premature birth, it is not possible in many cases. Preeclampsia is only one factor that leads to an increased risk of preterm labor, and thus, factors known to cause or be associated with preeclampsia are not necessarily causative factors or are associated with preterm labor.

Biomarkers

The disclosed methods relate to determining the presence or absence, or quantifying, of a biomarker. The biomarker is selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2.

Each biomarker used herein may be used alone or in combination with other biological preterm birth markers. In some assays, some or all of the biomarker proteins disclosed herein are used in combination. In addition, the biomarkers can be used with one or more other indicators of preterm birth, including Fetal Fibronectin (fn) testing, contractions, vaginal bleeding, fluid exudation from the vagina, increased vaginal discharge, back pain, and lower abdominal cramps.

Extracellular matrix protein 1(ECM 1); UniProtKB-Q16610

This gene encodes a 85kDa soluble protein involved in endochondrine formation (endochondridone formation), endothelial cell proliferation, angiogenesis (angiogenesis), and tumor biology. The proteins also interact with a variety of extracellular and structural proteins, which help maintain skin integrity and homeostasis. ECM1 may act as a "bio-gel" in a variety of tissues that aids in the organization and support of collagen.

This gene has been mentioned as having 4 alternatively spliced transcript variants encoding different isoforms. Any of the variants described herein can be used as biomarkers in accordance with the present disclosure. In some cases, variant 1 is detected. As shown in figure 1, the global mean concentration of ECM1 in all samples (collected between 19 and 37 weeks gestation) was lower in samples from individuals who underwent preterm birth than in samples that underwent term birth (p 0.025). This indicates a low expression of ECM1 in the sample, indicating that the individual is at risk of preterm birth.

This is further illustrated in fig. 2, where ECM1 was underexpressed in samples from subjects who underwent preterm birth at all sampling time points. In individuals suspected of being at risk of preterm birth, a lower amount of ECM1 may indicate that labor will occur in less than 12 weeks, less than 9 weeks, or less than 4 weeks.

Gamma-glutamyl hydrolase (GGH); UniProtKB-Q92820

GGH hydrolyzes the polyglutamic acid side chain of pteroyl polyglutamic acid, which gradually removes γ -glutamine residues from pteroyl poly- γ -glutamic acid to produce pteroyl- α -glutamic acid (folate) and free glutamic acid. It may play an important role in the bioavailability of dietary pteroylpolyglutamic acid and the metabolism of pteroylpolyglutamic acid and antifolates.

As shown in figure 4, the overall mean concentration of GGH in all samples (collected between 19 and 37 weeks gestation) was higher in samples from individuals who underwent preterm birth than in samples that underwent term birth. This indicates overexpression of GGH in the sample, indicating that the individual is at risk of preterm birth. As is particularly evident from FIGS. 5 and 6, GGH is particularly overexpressed in preterm samples within 1-4 weeks of gestation or at 32-37 weeks. This indicates that GGH is overexpressed in samples from 32 to 37 weeks of gestation, indicating that the individual is at risk for preterm birth. Conversely, low expression of GGH in samples taken prior to 32 weeks or within about 26 weeks of gestation may indicate that the individual is at risk of preterm birth. In individuals suspected of being at risk of preterm birth, an increased amount of GGH may indicate that delivery will occur within 4 weeks.

Laminin subunit gamma-2 (LAMC 2); UniProtKB-Q13753

LAMC2 is a heparin-binding protein that binds cells via high affinity receptors. Laminins are thought to mediate cell attachment, migration, and organization into tissues during embryonic development by interacting with other extracellular matrix components. Ladsin, a laminin variant containing the laminin gamma-2 chain, exerts cell-dispersing activity (cell-scattering activity) on a variety of cells, including epithelial cells, endothelial cells, and fibroblasts.

As shown in fig. 7, LAMC2 was higher in samples from individuals who underwent preterm birth than in samples that underwent term birth. This indicates overexpression of LAMC2 in the sample, indicating that the individual is at risk of preterm birth.

As shown in fig. 8, LAMC2 was overexpressed in preterm specimens less than 32 weeks gestation or in preterm specimens obtained less than 8 weeks before birth (fig. 9).

EGF extracellular matrix protein 1 containing fibronectin-like (EFEMP 1); UniProtKB-Q12805

The EFEMP1 protein binds to EGFR, the EGF receptor, and induces EGFR autophosphorylation (autophosphorylation) and downstream signaling pathway activation. It plays a role in cell adhesion and migration. It can be used as negative regulator of chondrocyte differentiation. In the olfactory epithelium (olfactary epithelium), it regulates glial cell migration, differentiation, and the ability of glial cells to support neurite outgrowth.

As shown in fig. 10, EFEMP1 was higher in samples from individuals who underwent preterm birth than in samples that underwent term birth. This indicates overexpression of EFEMP1 in the sample, indicating that the individual is at risk of preterm birth. As is also evident in fig. 11 and 12, EFEMP1 increased in preterm specimens at least 8 weeks prior to birth or 28 weeks gestation.

From a sample of individuals identified as being at risk for preterm birth, a significant upregulation of EFEMP1 may indicate that labor will be delivered within the next 1-4 weeks. From samples between 18-24 or 19-26 weeks gestation, down-regulation of EFEMP1 may indicate that the individual will experience preterm birth.

Pleiotropic growth factor (Pleiotrophin; PTN); UniProtKB-P21246

PTN is a secreted growth factor that induces neurite outgrowth and has mitogenic effects on fibroblasts, epithelial cells, and endothelial cells (PubMed:1768439, PubMed: 1733956). It binds to Anaplastic Lymphoma Kinase (ALK), which induces activation of the MAPK pathway, an important step in the regulation of PTN anti-apoptotic signaling and cell proliferation (PubMed: 11278720). It binds to cell surface target proteins via chondroitin sulfate (chondritinsulfate) groups (PubMed: 26896299). Upon binding, PTN inactivates the dephosphorylating enzyme activity of PTPRZ 1.

As shown in fig. 13, PTN was lower in samples of individuals who underwent preterm birth than in samples that underwent term birth. This shows that low expression of PTN in the sample may indicate that the individual is at risk of preterm birth. It is also evident in fig. 14 and 15, where PTN is reduced in samples taken from individuals who underwent preterm birth, within 32 weeks of gestation, or more than 4 weeks prior to birth. Over-expression of PTN in a sample may indicate that the individual will experience preterm birth within the next 1-4 weeks.

Fibrinogen alpha chain (FGA); UniProtKB-P02671

FGA is cleaved by the protease thrombin (thrombin) to produce monomers which polymerize with fibrinogen β (FGB) and fibrinogen γ (FGG) to form an insoluble fibrin matrix. Fibrin has a major function in the hemostasis (haemostasis) process, which is one of the major components of blood clots. In addition, it acts at an early stage of wound repair to stabilize the lesion and to guide cell migration during re-epithelialization (re-epithelialization). FGA was originally considered necessary for platelet aggregation based on in vitro studies of anticoagulation. However, subsequent studies have shown that its thrombus formation (thrombosis) in vivo is not absolutely necessary. FGA enhances the expression of P-Selectin (SELP) in activated platelets via the ITGB 3-dependent pathway. Maternal fibrinogen is a necessary condition for successful pregnancy. Fibrin deposition is also associated with infection, which can prevent IFNG-mediated bleeding. It may also promote immune responses through innate and T cell-mediated pathways.

As shown in fig. 16, FGA was higher in samples of individuals who underwent preterm birth than in samples that underwent term birth. This indicates that FGA is overexpressed in the sample, indicating that the individual is at risk of preterm birth. It is also evident in fig. 17 and 18 that overexpression of FGA in a sample may indicate that an individual may experience preterm birth, particularly when the sample is collected before 24 weeks gestation or after 32 weeks gestation. Overexpression of FGA may indicate that the individual may experience preterm birth within the next 1-4 weeks.

Low expression of FGA in samples collected between weeks 24 and 28 of gestation may indicate that the individual may experience preterm birth.

Pigment Epithelium-Derived Factor (Pigment epithelial-Derived Factor; PEDF); UniProtKB-P36955

PEDF is a neurotrophic protein (neurotrophin) that induces extensive neuronal differentiation in retinoblastoma (retinoblastoma) cells and is a potent inhibitor of angiogenesis. It has no serpin inhibitory activity since it does not undergo the S (stress) to R (relaxation) conformational transition characteristic of active serpins.

As shown in fig. 19, PEDF was higher in samples of individuals who underwent preterm birth than in samples that underwent term birth. This indicates that PEDF is overexpressed in the sample, indicating that the individual is at risk for preterm birth. It is also evident from figures 20 and 21 that overexpression of PEDF in a sample may indicate that the individual may experience preterm birth, regardless of the time of sampling. Overexpression of PEDF indicates that the individual may experience preterm birth within the next 1-4 weeks.

Particular methods disclosed herein relate to detecting biomarker values or levels of biomarkers. The term refers to the detection of a biomarker in a biological sample using any suitable analytical method, and indicates its presence, absence, absolute amount or concentration, relative amount or concentration, titer, amount, expression amount, ratio, or other measure of the biomarker in the corresponding sample. The exact nature of the value or amount depends on the particular design and composition of the particular assay used to detect the biomarker.

If a biomarker indicates that an individual is at risk of preterm birth, it may be over-or under-expressed as compared to a reference value or amount, or a biomarker indicative of or having a sign of term production. "upregulation," "overexpression," "increase," and related terms mean that the value or amount of a sample is greater than the value or amount (or range of values or amounts) of a biomarker, the latter typically being detected in a similar sample of an individual known to have experienced term delivery.

By "downregulating," "low expressing," "decreasing," and related terms, it is meant that the value or amount of a sample is less than the value or amount (or range of values or amounts) of a biomarker, the latter typically being detected in a similar sample of an individual known to have experienced term birth.

Biomarker overexpression or low expression may also refer to an amount or value that is considered "differentially expressed" or has a "difference" compared to the amount or value observed in individuals known to undergo term birth. Differential expression may also refer to the variation in the "normal" expression level of a biomarker.

The terms "differential gene expression" and "differential expression" are used interchangeably to represent a gene (or its corresponding protein expression product) that is activated to a higher or lower amount in a subject at risk of preterm birth relative to its activation in an individual known to have experienced term birth. The term also includes that the expression of a gene (or the corresponding protein expression product) is activated to higher or lower amounts in different stages of the same disease. It is also understood that the amount of nucleic acid or protein mass of differentially expressed genes may be activated or inhibited, or may be alternatively spliced (alternative splicing), to produce different polypeptide products. Such differences can be evidenced by a variety of variations, including mRNA quantity, surface expression, secretion, or other means of differentiating the polypeptide. Differential gene expression may include a comparison of expression between two or more genes or gene products thereof; or a comparison of the expression ratio between two or more genes or gene products thereof; or even a comparison of two different processed products of the same gene, which differs between individuals at risk of preterm birth or term birth. Differential expression includes both quantitative and qualitative differences in the temporal or cellular expression pattern of a gene or its expression product in individuals undergoing preterm and term delivery.

Results of the method

The methods disclosed herein are useful for identifying an individual as at risk for preterm birth, or for determining whether an individual is at risk for preterm birth or not. The method may also be used to predict the risk of preterm birth in an individual.

In some cases, the method may involve the step of recording the level of the biomarker. In some cases, the method may involve the step of communicating the levels of the biomarkers disclosed herein to a physician participating in the care of the subject. In some cases, the method also involves transmitting a biomarker reference level for comparison with the level of the biomarker of the individual. In some cases, the determined risk amount for preterm birth for the individual is communicated to a physician. For example, the risk amount may be expressed as a percentage (where 100% indicates that the individual is determined to experience preterm birth and 0% indicates that the individual is determined to experience term birth). Thus, some methods disclosed herein involve assigning a percentage value to an individual determined to have a risk amount. The method involves the step of communicating the percentage to a physician participating in the care of the individual.

The methods disclosed herein can be used to select individuals for treatment or other management. Particular methods disclosed herein relate to administering treatment to an individual identified as at risk for preterm birth.

Treatments useful in the methods disclosed herein include administration of progesterone, synthetic progesterone or progesterone analogs, one or more agents selected from progesterone or analogs thereof, tocolytics, corticosteroids, antibiotics, NSAIDs, or omega 3 fatty acids or derivatives thereof. The progesterone can be a synthetic progesterone, such as 17-a-hydroxyprogesterone caproate. The miscarriage preventing agent can be magnesium sulfate, indometacin, or nifedipine. The antibiotic may be erythromycin or penicillin. The NSAID may be indomethacin. The omega 3 fatty acid derivative may be docosahexaenoic acid (DHA).

Progesterone treatment may comprise administration of natural progesterone, or synthetic progestins (progestins), such as 17- α -hydroxyprogesterone caproate, progesterone may be P4 micronized (natural) progesterone, 17- α -hydroxyprogesterone caproate, known under the brand name Delalutin^TM、Proluton^TM、Proluton Depot^TMAnd Makena^TM. Natural micronized progesterone is similar to natural progesterone, as produced in the corpus luteum and placenta. The micronized progesterone can be used as an oral capsule, vaginal gel, or vaginal suppository. Synthetic progestins include medroxyprogesterone acetate (MPA, also known as storage medroxyprogesterone acetate (DMPA)) and norethindrone acetate (NETA). They are usually administered by injection. Known brand names for synthetic luteinizing hormones are (MPA) Provera^TM,Depo-Provera^TM,Depo-SubQProvera104^TM,Curretab^TM,Cycrin^TM,Farlutal^TM,Gestapuran^TM,Perlutex^TM,Veramix^TMand(NETA)Primolut-Nor^TM,Aygestin^TM,Gestakadin^TM,Milligynon^TM,Monogest^TM,Norlutate^TM,PrimolutN^TM,SH-420^TM,Sovel^TM,Styptin^TM. The micronized progesterone can be self-administered by the patient. Natural micronized progesterone is also known under the trade name Prometrium^TM,Utrogestan^TM,Endometrin^TMand Crinone^TMAlso disclosed are progestins, or 17- α -hydroxyprogesterone caproates for use in such methods, or progestins, or 17- α -hydroxyprogesterone caproates for use in the manufacture of medicaments for use in such methods.

Individuals identified as at risk for preterm birth may be treated with cervical cerclage. Cervical cerclage may also be referred to as cervical stiching. Cervical cerclage is used to treat cervical insufficiency or insufficiency, which shortens and opens the cervix too early during pregnancy. Cervical cerclage may involve a strong suture inside and around the cervix.

Any known form of cervical cerclage may be used. For example, the cervical cerclage may be McDonald cerclage, schrocard cerclage, or abdominal cerclage. Cervical cerclage may be particularly useful in individuals who are determined to have cervical insufficiency. Cervical insufficiency can be determined by transvaginal ultrasound scanning (transvascular ultrasound scan).

Alternatively, the treatment may comprise cervical pessary (cervical pessary). In some cases, the treatment may be arabinin Pessary (arabinin Pessary).

In some cases, the individual may be screened to receive an anti-miscarriage agent or a steroid, such as a corticosteroid. Miscarriage prevention agents can be used to prevent uterine contractions during preterm labor. Steroids may contribute to fetal lung development. The method may involve the step of administering to the subject an anti-miscarriage agent or a steroid. Miscarriage prevention agents and steroids have been used in women for uterine contractions. Examples of anti-abortion agents suitable for use in the present invention are magnesium sulfate, indomethacin, and nifedipine.

In some cases, the method is used to select an individual for further, periodic, or intensive monitoring. For example, the method may be used to determine that more samples should be obtained from the individual and, in the future, the presence or absence and/or amount of biomarkers. Further samples may be taken 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, or 36 weeks after the first sample. Further samples may be taken at 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, or 27 weeks of gestation. The method may involve further sampling every 1, 2, 3, 4, or 5 weeks during pregnancy.

In some cases, the individual may be treated with antibiotics. Antibiotics may be particularly useful in individuals with premature rupture of membranes (PPROMs). Suitable antibiotics include erythromycin and penicillin.

In some cases, the treatment may be the administration of an NSAID. NSAIDs can inhibit prostaglandins to reduce uterine contractions. The NSAID may be indomethacin. In some cases, the treatment may be an omega 3 fatty acid or a derivative of an omega 3 fatty acid. For example, the treatment may be DHA (docosahexaenoic acid).

Monitoring may include monitoring fetal distress (total stress), such as monitoring fetal heartbeat, monitoring fetal movement, or monitoring meconium.

Biomarker assay

The biomarkers disclosed herein are preferably protein biomarkers. Any method known in the art for detecting and/or quantifying proteins may be used.

The methods of the invention may be performed in vitro, ex vivo, or in vivo, or the product may be presented. The term "in vitro" refers to experiments under laboratory conditions or in culture, covering materials, biological substances, cells, and/or tissues, while the term "in vivo" refers to experiments and procedures covering organisms with intact multiple cells. "ex vivo" refers to something that exists or occurs outside of an organism, such as a human or animal body, which can be performed on a tissue (e.g., a whole organ) or cell of the organism.

The methods disclosed herein relate to determining protein expression. Protein expression can be measured by quantifying the amount of protein in a cell, tissue, or sample, or by observing the location of the protein within the cell and tissue.

In some cases, the immunoassay is used to detect a biomarker target in a sample from a subject. Immunoassays use antibodies or other entities with specific affinity for a target molecule in conjunction with a detectable molecule. In some cases, the antibody binds to a detectable molecule. The detectable molecule may refer to a label. When the antibody binds to the target molecule, the detectable molecule generates a detectable signal. The detectable signal may be a quantifiable signal. In some cases, an aptamer (aptamer) is used in place of, or in addition to, an antibody. Immunoassays include ELISA, immunoblotting, flow cytometry, and immunohistochemistry. In certain aspects described herein, the assay is an immunohistochemical assay. Such assays typically use antibodies, although other target-specific molecules, such as aptamers or other ligands, may also be used. Antibody arrays or protein chips may also be used.

The method is approved for use by regulatory agencies. The present method is an FDA approved method.

Antibodies

Antibodies that bind to the biomarkers of the invention are known. Given the technology now associated with monoclonal antibody technology, antibodies can be made against a large number of antigens.

The antigen-binding portion can be a portion of an antibody (e.g., a Fab fragment) or a portion of a synthetic antibody fragment (e.g., a single chain Fv fragment [ ScFv ]). Suitable Monoclonal Antibodies to the selected antigen can be prepared using known techniques, such as Monoclonal Antibodies A manual of techniques, HZola (CRC Press, 1988); and monoclonal hybridoma antibody: techniques and Applications (Monoclonal Antibodies: Techniques and Applications), J G R Hurrell (CRC Press, 1982). Chimeric antibodies were described by Neuberger et al, 1988, second Part of the eighth International Biotechnology Association, pages 792-799 (1988,8th International Biotechnology Symposium Part 2, 792-799).

Monoclonal antibodies (mAbs) are suitable for use in the methods of the invention and are a homogeneous population of antibodies that specifically target a single epitope on an antigen. Suitable monoclonal antibodies can be prepared using methods known in the art (see, e.g., forG; milstein, C. (1975). Continuous culture of fused cells secreting predetermined specific antibodies (Continuous cells of fused cells secreting antibodies of predetermined specificity), Nature 256(5517): 495; siegel DL (2002) Recombinant monoclonal antibody technology (regrbinant monoclonal antibody technology), Schmitz U, Versmold a, Kaufmann P, Frank HG (2000); for review of phage display: molecular tools for antibody production (phase display: a molecular tool for the generation of antibodies-a review.) planta.21 supplement a: S106-12. Helen e.chadd and stevenm.chamow; therapeutic antibody expression technology (Therapeutic antibody expression technology), Current Opinion in Biotechnology 12, No.2 (4/1/2001): 188-; McCafferty, j.; griffiths, a.; winter, g.; chiswell, d. (1990) phage antibodies: filamentous phages displaying antibody variable domains (phages: filamentous Phage displaying antibodies variable domains) Nature 348(6301): 552-554; monoclonal antibodies technical handbook (monoclonal antibodies: A manual of techniques), H Zola (CRC Press, 1988) and in monoclonal hybridoma antibodies: techniques and Applications (Monoclonal Antibodies: Techniques and Applications), JG R Hurrell (CRC Press, 1982) chimeric Antibodies were discussed by Neuberger et al, part II of the eighth International Biotechnology Association, 1988, page 792-799 (1988,8th International Biotechnology symposium part 2, 792-799).

Polyclonal antibodies are suitable for use in the methods of the invention. Preferably, it is a monospecific polyclonal antibody. Suitable polyclonal antibodies can be prepared using methods known in the art.

Antibody fragments, such as Fab and Fab2 fragments, can also be used or engineered as antibodies and antibody fragments. The fact that the Variable Heavy (VH) and Variable Light (VL) domains of antibodies are involved in antigen recognition was first confirmed in early protease digestion experiments. Further confirmation was by "humanization" of rodent antibodies. The variable domains of rodent origin are fused to constant domains of human origin, and the antibody so obtained retains the antigen specificity of the rodent parent antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

The antigen specificity is conferred by variable domains and is independent of constant domains, all of which are known from experiments in which bacteria express antibody fragments, and all contain one or more variable domains. Such molecules include Fab-like molecules (Better et al (1988) Science 240,1041); fv molecules (Skerra et al (1988) Science 240,1038); single chain fv (ScFv) molecules in which VH and VL partner domains are linked via a flexible polypeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sd. USA 85,5879), and single domain antibodies (dAbs) comprise an isolated V domain (Ward et al (1989) Nature 341, 544). A general overview of the techniques involved in the synthesis of antibody fragments and their retained specific binding sites can be found in Winter & Milstein (1991) Nature 349, 293-299.

The term "ScFv molecule" means a molecule in which VH and VL partner domains are covalently linked. For example, directly, via a polypeptide or via a flexible oligopeptide.

Fab, Fv, ScFv, and dAb antibody fragments can be expressed and secreted in E.coli, and therefore large quantities of the fragments can be easily produced.

The whole antibody, and the F (ab')2 fragment are "bivalent". The term "bivalent" means that the antibody has two antigen binding sites with the F (ab')2 fragment. In contrast, Fab, Fv, ScFv, and dAb fragments are monovalent, having only one antigen binding site. Synthetic antibodies that bind biomarkers can also be prepared using Phage display (phase display) techniques known in the art (see, e.g., Phage display reviews: molecular tools for antibody production (phase display: a molecular tool for the generation of antibodies-a review.; planta.21 supplement A: S106-12. Helene E. Chadd and Steven M. Chamow.; Phage antibodies: filamentous Phage displaying antibody variable domains. (phase of phase display variable domains); Nature 348(6301): 552-554).

In some preferred embodiments, the antibody is detectably labeled, or at least capable of detection. For example, the antibody may be a radioactive atom or a coloured molecule (chromophore) or a fluorescent molecule or a molecular label that can be easily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates, and radioactive labels. The antibody may be directly labeled with a detectable label, or may be indirectly labeled. For example, the antibody may be unlabeled and may be detected by another antibody that is itself labeled. Alternatively, the secondary antibody may bind biotin (biotin), which is bound with labeled streptavidin, and may be used to indirectly label the primary antibody.

One aspect disclosed herein is a complex of an antibody and a biomarker selected from the group consisting of ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC 2. The complex may further comprise a secondary, different antibody. The complex may further comprise a detectable moiety. The complexes may be present in a sample of cervicovaginal fluid. The complex may be isolated.

Detection and labelling

The methods disclosed herein relate to detecting and/or quantifying biomarkers.

In the methods disclosed herein, biomarkers (i.e., "targets") can be detected directly. That is, the target is detected by using an anti-target antibody.

Alternatively, the target may be detected indirectly. That is, the target can be detected using an anti-target antibody, which in turn can be detected using a secondary detectable anti-body. The secondary antibody is preferentially labeled. Suitable secondary antibodies may be antibody isoforms directed against animal species in which the primary antibody has been produced. For example, the secondary antibody can be an anti-mouse antibody that binds a mouse antibody. Methods using secondary antibodies may be more sensitive than direct detection methods, as multiple secondary antibodies bind to each primary antibody with signal amplification.

Suitable labels include enzymes such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, and luciferase, as well as colorimetric reagents including quantum dots, fluorophores, and chromophores. Suitable fluorophores include FITC, TRITC, Cy5, TexasRed, Alexa Fluor, and the like. The label may be a radioactive label.

There are a number of detectable enzyme substrates available for enzyme-labeled antibodies. These include chromogenic (chromogenic) detection systems such as horseradish peroxidase (HRP), pNNP, BCIP/NBT (5-bromo-4-chloro-3 '-indolylphosphate/nitro blue tetrazolium), TMB (tetramethylbenzidine), DAB (3, 3' -diaminediphenylbenzidine), o-phenylenediamine dihydrochloride (OPD), and 2,2 '-nitrilobis [ ethylbenzothiazoline-6-sulfonic acid ] (2, 2' -azinobis [ -ethylbenzothiazoline-6-sulfonic acid ]); ABTS), and chemiluminescent substrates, such as ECL (enhanced chemiluminescence) labels or Acridinium esters (Acridinium ester; AE).

The methods may involve the use of antibodies or antibody-derived binding agents, such as scFv or Fab fragments. Alternatively, in conjunction with antibodies, the method may involve the use of aptamers.

ELISA

In some cases, the target can be detected using enzyme-linked immunosorbent assay (ELISA). Target molecules (e.g., protein biomarkers disclosed herein) in a sample are attached to a surface and detected using specific antibodies. The target can be attached to the surface in a non-specific manner (via adsorption to the surface) or in a specific manner (using a specific capture reagent, such as an antibody). ELISA can be used to quantify the target in a sample. The surface may be a solid support such as a porous disk, a microbead, or a dipstick (dipstick).

Commercially available ELISA assays can be used. The ELISA may be an indirect-type ELISA, a sandwich-type ELISA, or a competitive-type ELISA.

ELISA involves the use of primary, capture antibodies to bind target molecules. Next, a secondary, detection antibody is added to the target molecule. The binding of the secondary antibody indicates the presence and/or amount of target.

The primary antibody may be bound to a solid support. Primary and secondary antibodies are not identical. Typically, the primary and secondary antibodies bind to different epitopes on the target molecule. In some cases, the secondary antibody binds to the complex of the primary antibody and the target, rather than the individual primary antibody or target without forming a complex. The secondary antibody may be labeled.

Immunostaining

In some aspects, the target is detected using immunoblotting or western blotting. In such methods, proteins in a sample may be separated according to their charge or size. It can be separated by electrophoresis. The isolated proteins were transferred to a thin film and stained with an antibody specific for the target. The antibody is then detected, either directly by binding the antibody to a detectable label, or indirectly by addition of a labeled secondary antibody.

Mass spectrometry

In some aspects, the methods disclosed herein relate to the detection and/or quantification of proteins using mass spectrometry. Mass spectrometry can use polypeptides with unique sequences of target proteins as surrogates for targets. The measurement is based on the peak mass and intensity of the protein, protein fragment, or portion of the polypeptide of interest. Prior to the measurement, a fixed amount of a substance as an internal standard was added to the original biomaterial and its peak intensity was measured. The target concentration in the raw biomaterial is calculated from the ratio of the peak intensity of the target to the peak intensity of the internal standard. A variety of known mass spectrometry methods, including MALDI-TOF (time of flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry, can be used to detect and/or quantify the biomarkers disclosed herein.

In vitro diagnostic agent and kit

One aspect of the present disclosure includes in vitro diagnostic methods, and in vitro diagnostic kits for using such methods. The kits described herein may include one or more antibodies, such as anti-biomarker antibodies or fragments thereof. The kit may be suitable for screening subjects at risk of preterm birth.

The kit may be adapted for point-of-care (point-of-care) in vitro diagnostic testing. It may be a kit for use in laboratory tests. The kit may include instructions for use, such as instructions or pamphlets. The instructions may comprise steps for performing any one or more of the methods described herein. The instructions may include steps for performing an immunochromatographic assay. Which may describe the test methods and recommendations for adjusting for different types of samples. It may provide methods and recommendations for optimizing the results obtained from the test, such as minimizing the signal-to-noise ratio.

The kit is suitable for immunochromatography tests. In some cases, in vitro diagnostic tests involve a lateral flow device (lateral flow device), or a "dipstick" test. In some cases, the kit includes a multi-well plate or other solid support that is pre-coated with a capture reagent, such as an anti-biomarker antibody.

The kit may additionally include a standard or control. The kit may additionally include buffers, diluents, or other reagents, such as stop buffers, sample preparation buffers, chromogenic reagents, streptavidin conjugates, substrates, or wash buffers.

The kit is suitable for use with a dry sample, a moist sample, a frozen sample, a fixed sample, a urine sample, a saliva sample, a tissue sample, a blood sample, or any other type of sample, including any of the types of samples disclosed herein.

The kit may comprise a device for taking or processing a vaginal fluid sample. The kit may comprise a vaginal fluid extraction buffer, such as a buffer containing about 50mM HEPES,150mM NaCl, 0.1% SDS,1mM EDTA,1mM Pefabloc SC 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF). The kit may comprise a sample collection device, such as a cotton swab (swab), a cervical-vaginal wick (cervicovaginalwick), a diaphragmlike device, a cervical aspirator, or a cell brush. The kit may comprise a container suitable for storing a vaginal fluid sample.

The cotton swabs suitable for the kit comprise foam cotton swabs (foam swabs), polyester cotton swabs (Dacron swabs), rayon cotton swabs (rayon swabs), flocking cotton swabs (flocked swabs) and cotton swabs. Suitable blister swabs include MW942(Sigma Swab Duo), polyurethane blister Swab (Catch All; Epicenter), and Culture Swab EZ polyurethane foam Swab (BD). Suitable dacron swabs include Deltalab Eurotuba 300263(Fisher scientific, UK), sterile G-in dacron tip plastic applicators (Solon, Clematic, Mycoplana), dacron swabs (Cardinal Health, Mgarura park, Illinois), and dacron swabs (Puritan Medical, Gilford, Mycoplana, USA). Suitable rayon swabs include BBL Cultur Schwab (Becton Dickinson, Oxford, UK) and MW167Suitable flocked swabs (nylon) include Seacliff packs, BD, COPAN. Suitable cotton swabs include sterile dry cotton swabs (eurotuba, Rubi, spain), cotton tipped cotton swabs (Falcon)^TMScrewCap Single SWUBE^TMApplicators, Becton Dickinson and Co., Spaxx, Maryland), Falcon^TMScrew Cap Single SWUBE^TMAn applicator (BD).

Suitable wicks for use in the kit include tampons, strips, or sponges, including ophthalmic PVA sponges (Eyetec)^TM,Network Medical Ltd.),Tear-Flo^TMA strip (Wilson opthalmic),sponges (XomedSurgical Products, Jacksville, Florida), Sno-strips (Akorn Inc., Abustaprils, los Angeles) and Polywicks (Polyfiltronics, Rockland, Mass., USA).

Suitable membrane-like devices for use in the kit include Instead softcups (Ultrafem), sterile gauze, or menstrual cups (softcups, euroferro, Netherlands, or the softcups, Instead inc., san diego, ca).

Suitable cervical aspirators include vaginal specimen aspirator (CarTika), or tuberculin (longtuboculin) syringe.

Particular kits disclosed herein comprise an antibody that binds to a preterm birth biomarker, and a device or buffer for taking or processing a vaginal fluid sample.

The antibody that binds to the preterm biomarker may be an anti-ECM 1 antibody, an anti-GGH antibody, an anti-LAMC 2 antibody, an anti-EFEMP 1 antibody, an anti-PTN antibody, an anti-FGA antibody, or an anti-PEDF antibody.

Also disclosed herein is a composition comprising vaginal fluid and an anti-ECM 1 antibody, an anti-GGH antibody, an anti-LAMC 2 antibody, an anti-EFEMP 1 antibody, an anti-PTN antibody, an anti-FGA antibody, or an anti-PEDF antibody.

Sampling method

The methods and agents described herein relate to the analysis of specific biomarkers in cervicovaginal fluid. Several known methods of sampling cervicovaginal fluid can be used in the present method.

The method may involve sampling with a cervical vaginal lavage. This involves rinsing the cervix and vagina with a wash buffer and collecting the fluid after rinsing to obtain a cervical-vaginal wash.

In some methods, a cervical-vaginal swab is used. Suitable swabs are well known in the art. Preferably, the swabs used in the methods and kits disclosed herein include foam swabs, polyester swabs, rayon swabs, flocking swabs, and cotton swabs. Suitable foam swabs include MW942(Sigma-Swab Duo), polyurethane foam swabs (Catch-All; Epicenter), and Curture SwabeZ polyurethane foam swabs (BD). Suitable dacron swabs include DeltalabEurotuba 300263(Fisher Scientific, UK), sterile G-in Dacron tip plastic applicators (Solon, Skoghegan, ME), Dacron swabs (Cardinal Health, McGraw Park, IL), and Dacron swabs (Puritanmedical, Guilford, ME, USA). Suitable rayon swabs include BBLCultureswab (Becton Dickinson, Oxford, UK) and MW167Suitable flocked swabs (nylon) include Seacliff packs, BD, COPAN. Suitable cotton swabs include sterile dry cotton swabs (Eurotuba, Rubi, Spain), cotton tipped cotton swabs (Falcon)^TMScrew Cap Single SWUBE^TMApplicators, Becton Dickinson and Co., Sparks, MD), Falcon^TMScrew Cap Single SWUBE^TMAn applicator (BD).

In other methods, cervicovaginal fluid is sampled with a wicking strip. Wicks suitable for use in the methods disclosed herein include tampons, strips, or sponges, including ophthalmic PVA sponges (Eyetec)^TM,Network Medical Ltd.),Tear-Flo^TMA strip (Wilson opthalmic),sponges (Xomed Surgical Products, Jacksonville, FL), Sno-strips (Akorn inc., Abita Springs LA) and Polywicks (Polyfiltronics, Rockland, MA, USA).

In other methods, a diaphragm-like device is used to sample the cervical-vaginal fluid. A suitable diaphragm-like device is placed over the cervix to collect the cervical-vaginal fluid and includes Instead SoftCup (Ultrafem), sterile gauze, or menstrual cup (SoftCup, euroferro, Netherlands, or the SoftCup, Instead inc., San Diego, CA).

The method also involves the use of a cervical aspirator, such as a vaginal specimen aspirator (CarTika), or a tuberculin syringe.

In some methods, cervicovaginal fluid is sampled with a cytobrush.

Particular kits disclosed herein comprise an antibody that binds to a preterm birth biomarker, and a device or buffer for taking or processing a vaginal fluid sample.

Control group

In some methods disclosed herein, the level of the biomarker is compared to a control group amount or reference value or amount.

In some cases, the control group may be a reference sample or reference data group. The reference set may be derived from one or more samples taken in advance from subjects known to have undergone preterm birth. Alternatively, the reference set may be derived from one or more samples taken in advance from subjects known to have undergone term delivery. The reference set may be a data set obtained by analyzing a reference sample.

The control group may be a positive control group in which the target molecule is known to be present or expressed in high amounts, or may be a negative control group in which the target molecule is known to be absent or expressed in low amounts.

The control group may be a sample or amount taken from a patient known to have undergone preterm or term birth. The value of the control group can be obtained by parallel analysis of the biomarkers using the individual samples to be tested. Alternatively, the values for the control group may be taken from a database or other previously obtained values.

Sample(s)

The methods disclosed herein relate to the detection of biomarkers in a sample taken from an individual or patient. The method may be carried out in vitro. Preferably, the method involves obtaining a sample from the subject. Thus, the present method is feasible, but not preferred, involving the step of obtaining a sample from an individual.

Preferably, the sample is a vaginal fluid sample, such as cervicovaginal (cervicovco-vaginal); cervicovaginal-vaginal) fluid (CVF) or cervical fluid (cervical fluid). Alternatively, the sample may be a blood sample, such as a whole blood, plasma, or serum sample, a lymph sample, a urine sample, or an amniotic fluid sample. The sample may be a protein sample derived from a vaginal fluid or cervicovaginal fluid sample, or a protein sample derived from a blood sample, such as a whole blood, plasma, or serum sample, a lymph sample, a urine sample, or an amniotic fluid sample.

The sample may be pre-treated. For example, the sample may have been previously contacted with one or more preservatives or buffers. The sample may be frozen, lyophilized, or dried.

Although the subject or patient can be a mammal, such as a cat, dog, horse, or ape, the subject is preferably a human. The terms "patient," "individual," and "subject" are used interchangeably herein.

The subject may be a female subject. The individual may become pregnant. The individual may be symptomatic or asymptomatic. Preferably, the subject is asymptomatic.

Symptomatic subjects may be subjects presenting with one or more symptoms of premature birth, such as contractions, especially regular contractions, back pain, including lower back pain, lower abdominal cramps, or the like menstrual cramps, fluid exudation from the vagina, the like flu symptoms, nausea, vomiting, increased pelvic or vaginal pressure, increased vaginal discharge, and/or vaginal bleeding.

Asymptomatic individuals may be asymptomatic for any symptoms or symptoms of preterm birth, which may or may not be indicative of preterm birth, such as back pain, including lower back pain, lower abdominal cramps, or similar menstrual cramps, fluid exudation from the vagina, similar flu symptoms, nausea, vomiting, increased pelvic or vaginal pressure, increased vaginal discharge, and/or vaginal bleeding. Typically, asymptomatic individuals do not develop any symptoms of premature birth.

In some cases, an individual may be suspected of being at high risk for preterm birth prior to taking a sample. For example, a sample may be obtained and/or the presence or amount of a biomarker may be determined, due to a suspected high risk of preterm birth for the individual. Individuals may be suspected of having a high risk of preterm birth due to a past history of preterm birth or abortion. Alternatively, or additionally, the subject may be suspected of having a high risk of preterm birth based on the results of their fetal fibronectin (fn) test, or based on symptoms such as contractions, vaginal bleeding, fluid exudation from the vagina, increased vaginal discharge, back pain, or lower abdominal cramps. Alternatively, or additionally, an individual may be considered to have a high risk of preterm birth due to the presence of one or more risk factors, such as diabetes, hypertension, having more than one infant, BMI (too high or too low), multiple vaginal infections, smoking, psychological stress, ethnic background, and socioeconomic status or income.

Samples may be taken weeks or months prior to birth, or prior to the edd. For example, a sample can be taken 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, or 36 weeks prior to birth. In some cases, samples are taken 1-4, 5-8, 9-12, or more than 12 weeks prior to birth.

Samples may be taken at some point prior to normal birth, with 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, or 36 weeks expected to be taken prior to birth, depending on the estimated 37-week period. In some cases, samples are taken 1-4, 5-8, 9-12, or more than 12 weeks prior to the expected normal birth date.

Alternatively, the sample may be taken about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, or about 9 months prior to the expected normal birth date.

Alternatively, the sample may be taken at 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, or 37 weeks of gestation.

Samples may be taken from 10 weeks to 13 weeks and 6 days of gestation, 14 weeks to 21 weeks and 6 days, 22 weeks to 25 weeks and 6 days, 26 weeks to 29 weeks and 6 days, 30 weeks to 33 weeks and 6 days, or more than 34 weeks. The first sample may be taken at about weeks 12-14. A second sample may be taken between 16-24 weeks.

The sample may be taken during the first, second, or third pregnancy. The first gestation period lasts from week 0 to 13 for another 6 days. The second gestation period lasts from week 14 to 27 plus 6 days. The third gestation period lasts from week 28 to birth.

Those skilled in the art will appreciate that it is difficult to accurately determine the number of weeks of pregnancy. Assessment of gestational weeks is a well known method in the art, and any of these can be used in the methods disclosed herein. For example, the number of weeks of pregnancy is usually estimated based on the date of the Last Menstrual Period (LMP). The number of weeks of gestation may depend on the date of the beginning of the last menstrual period. Alternatively, the number of weeks of gestation may be determined by the day of ovulation, if known. Typically, the day of ovulation is two weeks after the date of the start of the last menstruation. The length of the pregnancy may be determined by the delivery test. The labor check is usually performed between weeks 10 and 13 and day 6, depending on the day of the first day of the last menstruation.

Different biomarkers may be more appropriate for different sample times. For example, one biomarker may be useful in determining whether an individual is at risk of preterm birth in samples taken from individuals at an early stage, while a different biomarker may be useful in determining whether an individual is at risk of preterm birth in samples taken from individuals at a later stage.

In some cases, samples may be taken from an individual at multiple time points. For example, a first sample may be taken during a first pregnancy and a second sample may be taken during a second pregnancy. Multiple samples can be taken to determine trends or changes in biomarker expression. In some cases, a sample may be taken early in pregnancy, such as during the first pregnancy, to establish a baseline amount of control or biomarker for the individual.

Proteins and polypeptides

Although the methods of the invention may involve detection of full-length protein sequences, this is not always necessary. Alternatively, homologues, mutants, derivatives, isoforms, splice variants, or fragments of the full-length polypeptide may be detected.

Derivatives include variants of a given full-length protein sequence, and include naturally occurring allelic and synthetic variants that have substantial amino acid sequence identity to the full-length protein.

Protein fragments may be up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 150 amino acid residues in length. The minimum fragment length may be 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 30 amino acids, or a number of amino acids between 3 and 30.

The mutant may comprise at least one modification (e.g., addition, substitution, inversion, and/or deletion) compared to the corresponding wild-type polypeptide. The mutant may exhibit altered activity or properties, such as binding capacity.

Mutations can occur in any biomarker protein, and components containing such fragments can be used to modulate the activity of the mutant to restore, in whole or in part, the activity of the wild-type polypeptide.

Derivatives may also comprise natural variations or polymorphisms that may exist between individuals or between family members. All such derivatives are within the scope of the present invention. Purely by way of example, conservative substitutions may be found within such a polymorphism, and may be found within the following groups of amino acids:

alanine, serine, threonine;

glutamic acid and aspartic acid;

arginine and leucine;

aspartyl acid and glutamyl acid;

isoleucine, leucine, and valine;

phenylalanine, tyrosine, and tryptophan.

In the present specification, a biomarker may be any peptide, polypeptide, or protein whose amino acid sequence has a certain degree of sequence identity to one of the biomarker sequences or to a fragment of one of the sequences. A particular degree of sequence identity may be at least 60% to 100% sequence identity. More preferably, a particular degree of sequence identity can be one of at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments outlined above, many equivalent modifications and variations will be apparent to those skilled in the art when given the present disclosure. Accordingly, the exemplary embodiments of the invention, as set forth above, are intended to be illustrative only and not limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanation provided herein is intended only to improve the reader's understanding. The inventors do not wish to be bound by any such theoretical explanation.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, unless the context requires otherwise, including the following claims, the words "comprise" and "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. "about" is possible when related to a numerical value and means, for example, +/-10%.

Examples

Example 1

CVF (cervical vaginal fluid) samples were collected from pregnant women at 19-37 weeks gestation. A sterile bivalve speculum (bivalve speculum) is inserted into the patient's vagina. Double needle-tip swabs were placed in the posterior dome of the vagina for 30 seconds, followed by 1mL of cold CVF extraction buffer (50mM HEPES,150mM NaCl, 0.1% SDS,1mM EDTA,1mM Pefabloc SC 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF)). The sample was vortexed for 10 seconds, after which the swab was inverted and centrifuged at 1000Xg for 5 minutes. The swab was discarded and the sample tube was vortexed for 10 seconds, followed by centrifugation at 1000Xg for 5 minutes. The extracted CVF (supernatant) was aliquoted into tubes and stored at-80 ℃ until use.

Of the 200 CVF samples, 7 protein biomarkers (ECM1, GGH, LAMC2, EFEMP1, PTN, FGA, and PEDF) were tested, which were taken from 86 final term and preterm patients. Samples were collected in a longitudinal fashion during weeks 19-38 of gestation. The biomarker expression level in CVF samples was measured using commercial ELISA kits, namely PEDF (DuoSet, # DY1177-05, R & D Systems, Minneapolis, Minn., USA), ECM1(# ELH-ECM1-1, Raybiotech), GGH (# EH4206, Wuhan Feien Biotech, Inc.), LAMC2 SEC (# 083Hu, Cloud-clone), PTN (#23437, LSBIO), FGA (#11466, LSBIO), EFEMP1 MBS (# 178533, MyBioSource). The samples were run in a repeated fashion in standard 96-well plates along with a reference control and a standard protein of known concentration.

The ELISA procedure was performed according to the manufacturer's instructions. The overall procedure is described below:

the 96-well plates were coated with 100. mu.l of capture antibody at a concentration between 0.8-10. mu.g/ml in the coating buffer.

The plates were covered and incubated overnight at 4 ℃.

Add 300. mu.l of blocking solution to each well. Incubate for 60 minutes. The plates were washed three times with wash buffer and dried by patting the inverted plates on dry paper.

Add 100. mu.l of the gradient diluted protein standard and the appropriately diluted sample. The samples or standards were run in a repeated fashion and incubated at 37 ℃ for 90 min. The plates were washed three times with wash buffer and dried by patting the inverted plates on dry paper.

Add 100. mu.l of biotin bonding detection antibody, dilute with buffer reagent or appropriate buffer, and incubate at 37 ℃ for 1 hour. The plates were washed three times with wash buffer.

Mu.l of enzyme-linked streptavidin (streptavidin) was added, diluted with a buffer reagent or an appropriate buffer, and incubated at 37 ℃ for 60 minutes. The plates were washed three times with wash buffer and dried by patting the inverted plates on dry paper.

Add 100. mu.l of the appropriate substrate solution to each well. Incubate at 37 ℃ for up to 20 minutes, or until the desired color change is achieved.

The absorbance values were immediately read at the appropriate wavelength or 50. mu.l of "stop solution" was added. The plate was tapped to ensure thorough mixing. The absorbance at 450nm was measured, in accordance with the reference value of 540 nm.

Biomarker concentrations were determined from standard curves on each plate, the former being linear or 4 parameter log (4PL) standard curves. The final concentrations were normalized to the total protein concentration determined by the bicinchoninic acid assay (BCA assay).

Next, sample values and data were combined, and the results of term production and preterm production were compared and stratified according to 3 main methods:

the whole set (n 200) was evaluated, including the mean difference between term (n 136) and preterm (n 64) and confirmed p-values taken from student t-test analysis.

The gestational week samples are grouped according to the number of gestational weeks sampled.

Labor sample time is grouped by sample and day between births.

Statistical analysis

For statistical analysis, a two-tailed unpaired student's t-assay was performed using Microsoft Excel software, with a Confidence Interval (CI) of 0.95 and a P-value (P) of less than 0.05 considered significant. All numerical data, including error bars, represent mean +/-Standard Error of Mean (SEM).

Results and discussion

Biomarker quantification for 200 clinically derived samples confirmed the difference between term production and preterm samples. Further stratification of the sample enables the emphasis of potential time points in pregnancy which will help to better understand the pre-term risk basis.

ECM1 showed differential expression between all 200 term and preterm samples with a p-value of 0.0025 (fig. 1). Samples were stratified into different gestation periods and different sampling to time to delivery with differential expression remaining in the same direction (i.e., ECM1 was expressed less on average in all preterm samples, regardless of gestational age and time of delivery) (fig. 2 and 3). Since ECM1 is well known as a marker for several skin-related diseases and angiogenesis, its association with preterm labor is surprising.

GGH is differentially expressed between term and preterm samples. GGH expression was elevated on average in preterm versus term women samples (fig. 4). Interestingly, in the case of term delivery and preterm delivery, GGH expression levels gradually increased with increasing gestational age and decreasing time to delivery distance (fig. 5 and 6). GGH is not a well-known biomarker involved in immune pathways and extracellular matrix regulation.

LAMC2 was differentially expressed in all 200 term and preterm samples (fig. 7). Contrary to other markers, the difference was more evident on the last day before delivery (fig. 9). LAMC2 is involved in the epithelial turnover pathway and is known for its involvement in several dermatological indications. Interestingly, it has never been correlated with changes in the cervical-vaginal space.

EFEMP1 was also differentially expressed between all 200 term and preterm samples (fig. 10). More interestingly, however, there was a shift between increased expression and decreased expression of EFEMP1 throughout pregnancy between term and preterm birth samples. At the early time points of gestation time and time to delivery, EFEMP1 increased for the term specimen compared to the preterm specimen. However, this trend reversed late in gestation and during childbirth (fig. 11 and 12).

PTN was differentially expressed between all 200 term and preterm samples (fig. 13). The most significant differences in PTN expression profiles between term and preterm samples were observed at the earliest and latest time points from the time of delivery at the earliest and latest stages of pregnancy (fig. 14 and 15).

FGA was differentially expressed between all 200 term and preterm samples (fig. 16). The difference in FGA expression was more pronounced in late gestation and the last day before delivery (fig. 17 and 18). The proteinaceous form of FGA is involved in inflammatory and immune response pathways and may therefore be associated with premature birth cases triggered by these pathways.

PEDF was differentially expressed between all 200 term and preterm samples (fig. 19). PEDF continued to rise in the preterm samples in all stratification (fig. 20 and 21), indicating that it was a valid biomarker at any time point. PEDF is a protein closely related to angiogenesis and thus capable of remodeling tissues. The inventors hypothesized that their involvement in preterm birth is associated with cervical remodeling (cervical remodelling).

Today, the prediction of the risk of preterm birth in women is very limited. The two most common ways to predict risk profiles are based on medical history and cervical length. These methods fail to properly assess the risk of preterm birth in most women, even when used in combination. Therefore, if a tool for accurately predicting the premature birth risk of women can be provided, the tool becomes an important asset for the clinical pregnancy management, and the premature birth cases can be further reduced, and a large amount of medical expenses can be saved. The inventors herein demonstrate that individual biomarker tools can be used as a cornerstone. As can be seen, the kit of biomarkers ECM1, FGA, EFEMP1, GGH, PEDF, PTN, and LAMC2, will bind to the predictive value of individual biomarkers, as a highly accurate tool.

Example 2: in vitro assay

Ectocervical Ect1/E6E7(ATCC CRL-2614) and endocervical End1/E6E7(ATCC CRL-2615) cell lines were selected for biomarker in vitro studies under various cell stress conditions (hydrogen peroxide and LPS) according to the following steps:

_{2 2}HO processing

Ect1 and End1 cells were seeded in Keratinocyte Serum Free Medium (KSFM) supplemented with 0.1ng/ml EGF, 50. mu.g/ml BPE and 0.4mM CaCl on day 0₂. Increasing doses of H at day 2 to reach 70-80% confluency of cell growth₂O₂(50. mu.M, 100. mu.M, 200. mu.M, 400. mu.M) treatment of the cells. After 24 hours, the medium was collected and the biomarkers quantified using ELISA. Evaluation of H Using the MTT assay₂O₂Effects on cell viability and proliferation.

LPS treatment

Ect1 and End1 cells were seeded in KSFM supplemented with 0.1ng/ml EGF, 50. mu.g/ml BPEand 0.4mM CaCl on day 0₂. On day 1, the cell culture medium was removed and replaced with KSFM without growth factor supplement. Next, on day 2, cells were treated with increasing doses of LPS (10. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml). In that24 hours after LPS treatment, the medium was collected and the biomarkers quantified by ELISA.

Results and discussion:

the ectocervical Ect1/E6E7(ATCC CRL-2614) and endocervical End1/E6E7(ATCC CRL-2615) cell lines, both derived from normal cervical epithelial tissue, were selected for in vitro biomarker studies. End1 shows characteristics of simple columnar epithelial cells (simple columnar epithelial cells), while Ect1 shows characteristics of stratified squamous non-keratinizing epithelial cells (striified square nonkeratinizing epithelial cells). Since CVF is a fluid mixture derived from the vagina, cervix, and adjacent overlying fetal membranes (adjacencies), studying the secretory contents of Ect1 and End1 in the presence of different extracellular pressures can provide references and inferences about the local biochemical environment and physiological changes of the cervix during pregnancy.

Oxidative stress has been reported to play a major role in normal term and spontaneous preterm delivery (PTB) and many pregnancy complications, such as premature rupture of membranes (PPROM) and preeclampsia. Although the molecular mechanism is not clear, the role of oxidative stress in PTB can be attributed to various Reactive Oxygen Species (ROS) -mediated pathophysiological pathways such as inflammation, apoptosis, autophagy, aging, and alterations in collagen metabolism. The Menon et al study showed that in the fetal membranes of PTB and PPROM, the expression of oxidative stress markers such as F2-isoprostaglandin (F2-Isoprostanes) and OS-induced 3-nitrotyrosine-modified protein (3-NT) was significantly higher than that of fetal membranes of term birth. Meanwhile, both PTB and PPROM have higher amniotic fluid F2-isoprostane than term birth. The same group of researchers further concluded that oxidative stress-mediated apoptosis leads to proteolysis in fetal membranes, ultimately leading to membrane weakening and rupture in PPROMs. On the other hand, Heng et al show that as labor approaches, the expression of antioxidant enzymes, thioredoxin (thioredoxin), and SOD1, as well as the total antioxidant capacity of CVF, decreases significantly. It was concluded that labor is associated with increased oxidative stress and that antioxidant enzymes may be predictive of labor.

To induce oxidative stress in our system, increasing doses of H were used₂O₂Processing Ect1 and End1 detailsAnd carrying out cell culture for 24 hours. Evaluation of treatment H Using MTT assay₂O₂Cell viability and proliferation of cells (FIG. 22). The inventors found that H₂O₂Treatment induced a differential cellular response of Ect1 and End1 cells. Lower dose of H in Ect1 cells₂O₂(50. mu.M and 100. mu.M) did not affect cell viability. At 200 μ M H₂O₂In contrast, the cell density of the treated cells was reduced by 20% compared to the control untreated cells, although the reduction was not statistically significant. At 400 μ M H₂O₂In contrast, a significant decrease of-50% in cell viability of the treated cells was observed. In End1 cells, although 50. mu.M had no effect, 100. mu.M, 200. mu.M and 400. mu. M H₂O₂Results in a significant decrease in cell viability of-20%, -50%, and-90%, respectively.

The inventors used ELISA to treat H₂O₂The cell culture medium of (3) is subjected to quantification of biomarker expression. Interestingly, the biomarkers are in H₂O₂Differential expression was shown after treatment. In Ect1, FGA (FIG. 25), LAMC2 (FIG. 24), and EFEMP1 (FIG. 31) were expressed in the treatment of 200. mu.M and 400. mu.M H₂O₂After that, a significant reduction was found. When Ect1 was exposed to 400. mu. M H₂O₂After 24h in, ECM1 expression decreased (P)<0.05) (fig. 23). Expression of GGH in H₂O₂Remained relatively unchanged after treatment (fig. 26).

In End1 cells, 200. mu.M H₂O₂Significant changes in FGA and EFEMP1 expression were observed after treatment of the cells (fig. 25 and 32). The expression of ECM1 (FIG. 23), GGH (FIG. 26), and LAMC2 (FIG. 24) was similar to control untreated cells, albeit in H₂O₂And treating for 24 h. Tables 1 and 2 summarize the results as H₂O₂Changes in biomarker expression in Ect1 and End1 after treatment.

TABLE 1 Ect1 at H₂O₂Biomarker expression 24 hours after treatment

Fold change represents the mean of at least 4 independent experiments compared to control untreated cells. The results are expressed as fold change. + -. SEM. P <0.05, P < 0.005.

TABLE 2 End1 at H₂O₂Biomarker expression 24 hours after treatment

Fold change represents the mean of at least 3 independent experiments compared to control untreated cells. The results are expressed as fold change. + -. SEM. P < 0.005.

Similar to oxidative stress, inflammatory responses have long been associated with labor and term labor and labor at preterm birth. In most cases, PTB and PPROMs are closely related to intra-amniotic infection, intrauterine infection, and inflammation. Many experimental and clinical studies have suggested that pathophysiological pathways mediated by inflammatory mediators are the underlying cause of preterm labor and several pregnancy complications. The inflammation-mediated pathways include leukocyte activation, increased inflammatory and chemotactic interleukins, and collagenolysis by extracellular Matrix Metalloproteinases (MMPs). Such conditions ultimately lead to loss of membrane structural integrity, activation of the myometrium, and premature/early cervical remodeling, leading to PTB and PPROM. In addition, inflammatory markers, such as Interleukins (IL)1, 2, 6, and 8, tumor necrosis factor- [ TNF- ], and C-reactive protein [ CRP ], were evaluated as biomarkers for PTB.

In this study, the inventors treated Ect1 and End1 cells with different doses of Lipopolysaccharide (LPS) induced an inflammatory response in our system.

Treatment of 25 μ g/ml LPS in Ect1 cells resulted in a significant reduction in ECM1 expression in conditioned media (fig. 27). Furthermore, treatment with lower doses of LPS (10 μ g/ml and 25 μ g/ml) resulted in a statistically significant increase in expression of LAMC2 of more than 5-fold when compared to control untreated cells (fig. 28). The inventors also observed a slight increase in GGH (fig. 29) and FGA (fig. 30) expression in conditioned medium of Ect1 cells treated with three different doses of LPS, however, this increase was not statistically significant.

In End1 cells, similar to the Ect1 cells, treatment of LPS caused a small reduction in ECM1 expression in conditioned media (fig. 27), although this reduction was not statistically significant. In End1 conditioned medium, all three high and low doses of LPS (10. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml) resulted in an-8-fold increase in LAMC2 expression (FIG. 28). In contrast, the expression levels of GGH (fig. 29) and FGA (fig. 30) were down-regulated after LPS treatment in End1 conditioned medium. Changes in biomarker expression following LPS treatment are summarized in tables 3 and 4.

TABLE 3 biomarker expression of Ect1 after 24 hours of LPS treatment

Fold change represents the mean of at least 4 independent experiments compared to control untreated cells. The results are expressed as fold change. + -. SEM. P < 0.05.

TABLE 4 biomarker expression by End1 after 24 hours of LPS treatment

Fold change represents the mean of at least 4 independent experiments compared to control untreated cells. The results are expressed as fold change. + -. SEM. P <0.05, P < 0.005.

Reference to the literature

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