Pregnancy clock proteins for predicting due date and time to birth

BACKGROUND

Accurately assigning Estimated Due Date (EDD) and/or Time To Birth (TTB) early in prenatal care is among the most important results of evaluation and history taking. This information is vital for timing of appropriate obstetric care; scheduling and interpretation of certain antepartum tests; determining the appropriateness of fetal growth; and designing interventions to prevent preterm births, postterm births, and related morbidities. A consistent and exacting approach to accurate dating is also a research and public health imperative because of the influence of dating on investigational protocols and vital statistics.

Traditionally, determining the first day of the Last Mentstrual Period (LMP) is the first step in establishing the EDD. By convention, the EDD is 280 days after the first day of the LMP. Because this practice assumes a regular menstrual cycle of 28 days, with ovulation occurring on the 14th day after the beginning of the menstrual cycle, its accuracy is affected by factors that include inaccurate recall of the LMP, irregularities in cycle length, or variability in the timing of ovulation. Obstetric ultrasonography (US) is routinely used to determine fetal gestational age and aid in assigning EDD. If the patient is unsure of her LMP, dating of EDD based on first trimester US considered more reliable than second trimester or third semester US.

Both LMP and/or ultrasound are population-based estimates for a normal pregnancy and the accuracy of these methods varies significantly. Current clinical practice utilizing these methods is accurate in making a due date prediction that falls within plus or minus five days of the actual due date for term deliveries only about 35% of the time. In addition, 15% of predictions made under current clinical practice fall on or outside of 14 days before or after the actual due date for term deliveries. More accurate dating of pregnancy is needed to improve outcomes and is a research and public health imperative. The present invention addresses this need by providing an Estimated Due Date (EDD) molecular predictor (EDDmp) and/or Time To Birth (TTB) molecular predictor (TTBmp) that incorporates molecular information from proteins listed in Tables 1-27 into the estimation of pregnancy due date and/or time to birth with much higher accuracy than methods utilized as part of current clinical practice. Related advantages are provided as well.

SUMMARY

The present invention provides compositions and methods for due date and time to birth prediction for a pregnancy with significantly higher accuracy than current clinical methods. The compositions and methods for due date and time to birth prediction for a pregnancy can also identify those pregnancies, with high accuracy, that will deliver earlier than the official EDD as derived from LMP and/or US dating. Accordingly, the present invention provides an improved process that applies the discoveries described herein to enable, inter alia, a new and useful process for estimating the due date of a pregnant female, subsequently referred to as the Estimated Due Date (EDD) and/or estimating time to birth (TTB) with much higher accuracy than currently practiced clinical methods.

Each of the proteins, peptides and clinical variables disclosed herein as components of pairs, ratios and/or reversal pairs serve as biomarkers for determining the EDD, predicting gestational age at birth (GAB), predicting time to birth (TTB), either individually, in ratios, reversal pairs or in panels of biomarkers/reversal pairs.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the composition further comprises AACT_EIGELYLPK.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the composition further comprises AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, and FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, to determine the EDD for said pregnant female. In some embodiments, the pregnant female is nulliparous. In additional embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, and FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, to determine the EDD for said pregnant female. In some embodiments, the pregnant female is nulliparous. In additional embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, and FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, and FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, to determine the TTB for said pregnant female. In some embodiments, the pregnant female is nulliparous. In additional embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a reversal value of a biomarker pair selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, and FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, wherein said pair of biomarkers exhibits a change in a reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a biomarker pair selected from the group consisting of ADA12_FGFGGSTDSGPIR and PAEP_HLWYLLDLK, PAEP_HLWYLLDLK and PRG4_GLPNVVTSAISLPNIR, IBP4_Q.CHPALDGQR and PAEP_HLWYLLDLK, and FETUA_FSVVYAK and IBP4_Q.CHPALDGQR, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the composition further comprises AACT_EIGELYLPK.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the composition further comprises AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, to determine the EDD for said pregnant female. In some embodiments, the pregnant female is nulliparous. In additional embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, to determine the EDD for said pregnant female. In some embodiments the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a reversal value of a biomarker pair consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in a reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the pregnant female is nulliparous. In additional embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a biomarker pair consisting of FETUA_HTLNQIDEVK, PRG4_GLPNVVTSAISLPNIR or KNG1_DIPTNSPELEETLTHTITK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the pregnant female is nulliparous. In additional embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises measuring AACT_EIGELYLPK.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, and CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, and CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, and CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers selected from the group consisting of ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, and CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a reversal value of a biomarker pair selected from the group consisting of ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, and CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, wherein said pair of biomarkers exhibits a change in a reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a biomarker pair selected from the group consisting of ADA12_FGFGGSTDSGPIR and CRIS3_YEDLYSNCK, CO5_TLLPVSKPEIR and ADA12_FGFGGSTDSGPIR, AFAM_HFQNLGK and AACT_EIGELYLPK, ALS_IRPHTFTGLSGLR and PCD12_AHDADLGINGK, VTNC_GQYCYELDEK and PCD12_AHDADLGINGK, and CRIS3_YEDLYSNCK and TETN_LDTLAQEVALLK, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a reversal value of a biomarker pair consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in a reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a biomarker pair consisting of ADA12_FGFGGSTDSGPIR, PCD12_AHDADLGINGK, CD14_LTVGAAQVPAQLLVGALR or CRIS3_YEDLYSNCK and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In additional embodiments, the biological sample is blood and the gestational age at blood draw (GABD) is from 23 0/7 weeks and 28 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers selected from the group consisting of B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers selected from the group consisting of B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers selected from the group consisting of B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers selected from the group consisting of B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a reversal value of a biomarker pair selected from the group consisting of B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, wherein said pair of biomarkers exhibits a change in a reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a biomarker pair selected from the group consisting of B2MG_VEHSDLSFSK and FGFR1_IGPDNLPYVQILK, GELS_TASDFITK and FGFR1_IGPDNLPYVQILK, LIRB5_KPSLLIPQGSVVAR and FA9_SALVLQYLR, B2MG_VEHSDLSFSK and CHL1_VIAVNEVGR, and CHL1_VIAVNEVGR and IGF2_GIVEECCFR, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of CHL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of CHL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers consisting of CHL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the estimated due date (EDD) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers consisting of CRL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, to determine the EDD for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a reversal value for a pair of biomarkers consisting of CRL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method of determining the time to birth (TTB) for a pregnant female, the method comprising measuring in a biological sample obtained from said pregnant female a pair of biomarkers consisting of CRL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, to determine the TTB for said pregnant female. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the determination further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a reversal value of a biomarker pair consisting of CRL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in a reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, the method further comprises calculation of Inverse Parity as 1/(Parity 0.5).

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in a biomarker pair consisting of CHL1_VIAVNEVGR, FGFR1_IGPDNLPYVQILK or FA9_FGSGYVSGWGR and one of the biomarkers listed in Tables 1-27, wherein said pair of biomarkers exhibits a change between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks. In some embodiments, method further comprises calculation of Inverse Parity as 1/(Parity−0.5).

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers selected from the group consisting of the biomarker pairs listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the composition comprises AACT_EIGELYLPK.

In one embodiment, the present invention provides a composition comprising two or more pairs of isolated biomarkers selected from the group consisting of the biomarker pairs listed in Tables 1-27, wherein said pairs of biomarkers exhibit a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In some embodiments, the composition comprises AACT_EIGELYLPK.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in reversal value of two or more biomarker pairs selected from the group consisting of the biomarker pairs listed in Tables 1-27, wherein said pair of biomarkers exhibit a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention further provides a method for prediction of gestational age at birth (GAB).

In a further embodiment, the present invention provides a method for estimating gestational age (GA).

In one embodiment, the present invention further provides a method for prediction of time to birth (TTB).

In some of the embodiments, the methods have an accuracy of 60% or more for predicting the EDD within plus or minus 5 days of the actual due date (DD).

In additional embodiments, the methods comprise measuring AACT_EIGELYLPK.

In additional embodiments, the methods comprise calculation of Inverse Parity as 1/(Parity−0.5).

In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 18 0/7 weeks through 22 6/7 weeks.

In some embodiments, the biological sample is obtained at a gestational age at blood draw (GABD) from 23 0/7 weeks through 28 6/7 weeks.

In some embodiments, the measuring comprises mass spectrometry (MS). In one embodiment, the measuring further comprises measuring surrogate peptides of said biomarkers in the biological sample obtained from said pregnant female. In one embodiment, the measuring of surrogate peptides of said biomarkers further comprises measuring stable isotope labeled standard peptides (SIS peptides) for each of the surrogate peptides.

In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, and serum. In one embodiment, the biological sample is serum.

In some embodiments, the measuring comprises an assay that utilizes a capture agent. In one embodiment, the measuring comprises an assay that utilizes a capture agent selected from the group consisting of and antibody, antibody fragment, nucleic acid-based protein binding reagent, small molecule or variant thereof. In one embodiment, the measuring comprises an assay selected from the group consisting of enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Kinetic plot of the protein ratio of CATD/TENX over the a Gestational Age at Blood Draw (GABD) window of 140 to 153 shows an AUC of 82% in separating those subjects that gave birth significantly earlier (i.e. before 270 days) than the population average of 280 days.

FIG. 2 depicts a conditional inference tree for the prediction of a subject TTB's difference from the median TTB.

DETAILED DESCRIPTION

The present disclosure is based, generally, on the discovery that certain proteins and peptides in biological samples obtained from a pregnant female are differentially expressed in pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. The present disclosure is further based, generally, on the discovery that certain proteins and peptides in biological samples obtained from a pregnant female are differentially expressed in pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

The present disclosure is further specifically based, in part, on the unexpected discovery that pairs of biomarkers disclosed herein can be utilized in methods of estimating the due date of a pregnant female, subsequently referred to as the Estimated Due Date (EDD) and/or estimating time to birth (TTB). The present disclosure is further specifically based, in part, on the unexpected discovery that pairs of biomarkers disclosed herein can be utilized in methods of estimating the due date of a pregnant female, subsequently referred to as the EDD. Furthermore, each of the proteins, peptides and clinical variables disclosed herein as components of pairs, ratios and/or reversal pairs serve as biomarkers for determining the EDD, predicting gestational age at birth (GAB), predicting time to birth (TTB), estimating gestational age (GA) either individually, in ratios, reversal pairs or in panels of biomarkers/reversal pairs. Furthermore, the compositions and methods described herein comprise each of the proteins corresponding to the peptide biomarkers disclosed herein can serve as a component of pairs, ratios and/or reversal pairs for determining the EDD, predicting gestational age at birth (GAB), predicting time to birth (TTB), estimating gestational age (GA) either individually, in ratios, reversal pairs or in panels of biomarkers/reversal pairs. In addition, the compositions and methods described herein comprise surrogate peptides for each of the proteins corresponding to the peptide biomarkers disclosed herein can serve as a component of pairs, ratios and/or reversal pairs for determining the EDD, predicting gestational age at birth (GAB), predicting time to birth (TTB), estimating gestational age (GA), either individually, in ratios, reversal pairs or in panels of biomarkers/reversal pairs.

The present disclosure is further specifically based, in part, on the unexpected discovery that pairs of biomarkers disclosed herein can be utilized in methods of estimating the time to birth of a pregnant female, subsequently referred to as the Time To Birth (TTB). The present disclosure is further specifically based, in part, on the unexpected discovery that reversal values of pairs of biomarkers disclosed herein can be utilized in methods of estimating the due date of a pregnant female, subsequently referred to as TTB.

The present invention provides an improved process that applies the aforementioned discoveries to enable a new and useful process for estimating the due date of a pregnant female, subsequently referred to as the Estimated Due Date (EDD) and/or estimating time to birth (TTB) with much higher accuracy than currently practiced clinical methods.

The concepts of EDD and TTB are directly related and a skilled person can adjust the methods used to determine EDD to determine TTB and vice versa. Accordingly, the terms estimated due date (EDD) and time to birth (TTB) are used interchangeably in the context of predictors for DD. The EDD can be used to predict TTB and vice-versa. Explicitly, if the estimated gestational age of a pregnancy is X at the time of blood draw then TTB can be estimated from EDD as follows: TTB=EDD−X. And DD can be estimated from a TTB predictor as follows: EDD=X+TTB, where the units used are days.

The proteins and peptides disclosed herein as components of pairs, ratios and/or reversal pairs serve as biomarkers for determining the EDD, predicting gestational age at birth (GAB), predicting time to birth (TTB), either individually, in ratios, reversal pairs or in panels of biomarkers/reversal pairs.

A reversal value is the ratio of the relative peak area of an up regulated biomarker over the relative peak area of a down regulated biomarker and serves to both normalize variability and amplify diagnostic signal. The invention lies, in part, in the selection of particular biomarkers that, when paired together, can accurately determine the EDD and/or TTB based on pairs of biomarkers. Accordingly, it is human ingenuity in selecting the specific biomarkers that are informative upon being paired, for example, in novel reversals that underlies the present invention.

The disclosure provides biomarker reversal pairs and associated panels of reversal pairs, methods and kits for determining the EDD and/or TTB in a pregnant female.

In addition to the specific biomarkers identified in this disclosure, for example, by name, sequence, or reference, the invention also contemplates use of biomarker variants that are at least 90% or at least 95% or at least 97% identical to the exemplified sequences and that are now known or later discovered and that have utility for the methods of the invention. These variants may represent polymorphisms, splice variants, mutations, and the like. In this regard, the instant specification discloses multiple art-known proteins in the context of the invention and provides exemplary peptide sequences that can be used to identify these proteins. However, those skilled in the art appreciate that additional sequences or other information can easily be identified that can provide additional characteristics of the disclosed biomarkers and that the exemplified references are in no way limiting with regard to the disclosed biomarkers.

As described herein, various techniques and reagents find use in the methods of the present invention. Suitable samples in the context of the present invention include, for example, blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, and serum. In a particular embodiment, the biological sample is serum. As described herein, biomarkers can be detected through a variety of assays and techniques known in the art. As further described herein, such assays include, without limitation, mass spectrometry (MS)-based assays, antibody-based assays as well as assays that combine aspects of the two.

Protein biomarkers that are components of reversal pairs described herein include, for example, Cathepsin D (CATD) and Tenascin X (TENX).

In some embodiments, the invention provides a method of determining the EDD for a pregnant female, the method comprising measuring in a biological sample obtained from the pregnant female a reversal value for the biomarkers CATD and TENX.

The invention methods also contemplate measuring surrogate peptides of the biomarkers CATD and TENX. The biomarkers of the invention, their surrogate peptides and the corresponding stable isotope labeled standard peptides (SIS peptides) can be used in methods of determining the EDD for a pregnant female. In some embodiments, the SIS peptides correspond to surrogate peptides of the isolated biomarkers selected from the group consisting of CATD and TENX.

In some embodiments, the invention provides a pair of isolated biomarkers CATD/TENX, wherein the pair of biomarkers exhibits a higher ratio in pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers selected from the group consisting of the biomarker pairs listed in Table 1, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a pair of surrogate peptides of a pair of biomarkers selected from the group consisting of the biomarker pairs listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In one embodiment, the present invention further provides stable isotope labeled standard peptides (SIS peptides) corresponding to each of the surrogate peptides.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in reversal value for a pair of biomarkers selected from the group consisting of the biomarker pairs listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in reversal value of two or more biomarker pairs selected from the group consisting of biomarker pairs listed in Tables 1-27, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In some embodiments, the sample is obtained between 18 and 21 weeks of GABD. In further embodiments, the sample is obtained between 23 and 28 weeks of GABD. In some embodiments, the sample is obtained between 18 and 28 weeks of GABD. In some embodiments, the sample is obtained between 18 and 36 weeks of GABD. In further embodiments the sample is obtained between 19 and 21 weeks of GABD. In some embodiments, the sample is obtained between 20 and 22 weeks of GABD. In some embodiments, the sample is obtained between 21 and 23 weeks of GABD. In further embodiments, the sample is obtained between 22 and 24 weeks of GABD. In additional embodiments, the sample is obtained between 23 and 25 weeks of GABD. In some embodiments, the sample is obtained between 24 and 26 weeks of GABD. In further embodiments, the sample is obtained between 25 and 27 weeks of GABD. In additional embodiments, the sample is obtained between 26 and 28 weeks of GABD. In some embodiments, the sample is obtained between 27 and 29 weeks of GABD. In further embodiments, the sample is obtained between 28 and 30 weeks of GABD. In additional embodiments, the sample is obtained between 29 and 31 weeks of GABD. In some embodiments, the sample is obtained between 30 and 32 weeks of GABD. In further embodiments, the sample is obtained between 31 and 33 weeks of GABD. In additional embodiments, the sample is obtained between 32 and 34 weeks of GABD. In some embodiments, the sample is obtained between 33 and 35 weeks of GABD. In further embodiments, the sample is obtained between 34 and 36 weeks of GABD. In additional embodiments, the sample is obtained between 18 and 21 weeks of GABD.

In addition to the specific biomarkers, the disclosure further includes biomarker variants that are about 90%, about 95%, or about 97% identical to the exemplified sequences. Variants, as used herein, include polymorphisms, splice variants, mutations, and the like. Although described with reference to protein biomarkers, changes in reversal value can be identified in protein or gene expression levels for pairs of biomarkers.

The compositions and methods of the invention also can include clinical variables, including but not limited to, maternal characteristics, medical history, past pregnancy history, and obstetrical history. Such additional clinical variables can include, for example, previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortions, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, parity, nulliparity, placental abnormalities, cervical and uterine anomalies, short cervical length measurements, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low prepregnancy weight, low or high body mass index, diabetes, diabetes mellitus, chronic diabetes, chronic diabetes mellitus, chronic hypertension, urogenital infections (i.e. urinary tract infection), asthma, anxiety and depression, asthma, hypertension, hypothyroidism, high body mass index (BMI), low BMI, BMI. Demographic risk indicia for preterm birth can include, for example, maternal age, race/ethnicity, single marital status, low socioeconomic status, maternal age, employment-related physical activity, occupational exposures and environment exposures and stress. Further clinical variables can include, inadequate prenatal care, cigarette smoking, use of marijuana and other illicit drugs, cocaine use, alcohol consumption, caffeine intake, maternal weight gain, dietary intake, sexual activity during late pregnancy and leisure-time physical activities. (Preterm Birth: Causes, Consequences, and Prevention, Institute of Medicine (US) Committee on Understanding Premature Birth and Assuring Healthy Outcomes; Behrman R E, Butler A S, editors. Washington (DC): National Academies Press (US); 2007). Additional clinical variables useful for as markers can be identified using learning algorithms known in the art, such as linear discriminant analysis, support vector machine classification, recursive feature elimination, prediction analysis of microarray, logistic regression, CART, FlexTree, LART, random forest, MART, and/or survival analysis regression, which are known to those of skill in the art and are further described herein.

The present disclosure describes and exemplifies various models and corresponding biomarkers that perform at high levels of accuracy and precision in predicting the actual due date. It will be understood by those of skill in the art, that other models are known in the art that can be used to practice the claimed inventions and that the performance of a model can be evaluated in a variety of ways, including, but not limited to accuracy, precision, recall/sensitivity, weighted average of precision and recall. Models known in the art include, without limitation, linear discriminant analysis, support vector machine classification, recursive feature elimination, prediction analysis of microarray, logistic regression, CART, FlexTree, LART, random forest, MART, and/or survival analysis regression.

In some embodiments, performance of a model can be evaluated based on accuracy, which can be described as the difference between the EDD and the actual due date. For example, accuracy can be expressed as the percentage of time, for example, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 80% or more that a model provides an EDD that falls within a certain range of days, for example, +/−10 days, +/−9 days, +/−8 days, +/−7 days, +/−6 days, +/−5 days, +/−4 days, +/−3 days +/−2 days, +/−1 day of the actual due date. In one embodiment, accuracy can be described by noting that the EDD or TTB predictor is accurate to within +/−5 days of the actual DD or TTB for a term pregnancy at least 60% of the time.

The present disclosure is based in part on the surprising discovery that the selection of certain biomarkers and/or clinical variables enables determining EDD and/or TTB at a significantly higher level of accuracy and precision compared to current clinical practice, which is accurate in making a due date prediction that falls within +/−5 days of the actual due date only about 35% of the time. In contrast, the present invention provides and exemplifies compositions and methods that enable a prediction time to birth or due date that falls within plus or minus five days of the actual time to birth or due date about 60% of the time.

It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a biomarker” includes a mixture of two or more biomarkers, and the like.

The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.

As used herein, the term “panel” refers to a composition, such as an array or a collection, comprising one or more biomarkers. The term can also refer to a profile or index of expression patterns of one or more biomarkers described herein. The number of biomarkers useful for a biomarker panel is based on the sensitivity and specificity value for the particular combination of biomarker values.

As used herein, and unless otherwise specified, the terms “isolated” and “purified” generally describes a composition of matter that has been removed from its native environment (e.g., the natural environment if it is naturally occurring), and thus is altered by the hand of man from its natural state so as to possess markedly different characteristics with regard to at least one of structure, function and properties. An isolated protein or nucleic acid is distinct from the way it exists in nature and includes synthetic peptides and proteins.

The term “biomarker” refers to a biological molecule, a fragment of a biological molecule, or a clinical variable the change and/or the detection of which can be correlated with a particular physical condition or state. The terms “marker” and “biomarker” are used interchangeably throughout the disclosure. For example, the biomarkers of the present invention are associated with a discrimination power between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. Such biomarkers include any suitable analyte, but are not limited to, biological molecules comprising nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as surrogates for biological macromolecules and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also encompasses portions or fragments of a biological molecule, for example, peptide fragment of a protein or polypeptide that comprises at least 5 consecutive amino acid residues, at least 6 consecutive amino acid residues, at least 7 consecutive amino acid residues, at least 8 consecutive amino acid residues, at least 9 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 11 consecutive amino acid residues, at least 12 consecutive amino acid residues, at least 13 consecutive amino acid residues, at least 14 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 5 consecutive amino acid residues, at least 16 consecutive amino acid residues, at least 17 consecutive amino acid residues, at least 18 consecutive amino acid residues, at least 19 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 21 consecutive amino acid residues, at least 22 consecutive amino acid residues, at least 23 consecutive amino acid residues, at least 24 consecutive amino acid residues, at least 25 consecutive amino acid residues, or more consecutive amino acid residues.

As used herein, the term “surrogate peptide” refers to a peptide that is selected to serve as a surrogate for quantification of a biomarker of interest in an MRM assay configuration. Quantification of surrogate peptides is best achieved using stable isotope labeled standard surrogate peptides (“SIS surrogate peptides” or “SIS peptides”) in conjunction with the MRM detection technique. A surrogate peptide can be synthetic. An SIS surrogate peptide can be synthesized with heavy labeled for example, with an Arginine or Lysine, or any other amino acid at the C-terminus of the peptide to serve as an internal standard in the MRM assay. An SIS surrogate peptide is not a naturally occurring peptide and has markedly different structure and properties compared to its naturally occurring counterpart. For any of the embodiments described herein, the biomarkers can be quantified by measuring surrogate peptides.

In some embodiments, the invention provides a method of separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring in a biological sample obtained from the pregnant female a ratio for at least a pair of biomarkers consisting of CATD/TENX to determine the EDD for said pregnant female, wherein a higher ratio indicates a greater likelihood of delivery before 270 days.

In some embodiments, the invention provides a method of separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring in a biological sample obtained from the pregnant female a ratio for at least a pair of biomarkers consisting of CATD/TENX to determine the EDD for said pregnant female, wherein a lower ratio indicates a greater likelihood of delivery on or after 280 days.

The term “clock protein” as used herein, refers to biomarkers that provide information on the due date of a pregnant subject, the state of development and/or age of a fetus or the progress through pregnancy. There are a number of important ways that these biomarkers can be advantageously used in assessing development including, for example, (1) for the prediction of gestational age at birth or time to birth (TTB) from the moment the blood is drawn to deliver and (2) for prediction of the gestational age at the time blood is drawn. In addition, clock proteins can serve to normalize component peptides in signatures to improve predictive performance or to select appropriate biomarkers and/or classifiers.

As used herein, the term “reversal” refers to the ratio of the measured value of an upregulated analyte over that of a down-regulated analyte. In some embodiments, the analyte value is itself a ratio of the peak area of the endogenous analyte over that of the peak area of the corresponding stable isotopic standard analyte, referred to herein as: response ratio or relative ratio.

As used herein, the term “reversal pair” refers to biomarkers in pairs that exhibit a change in value between the classes being compared. The detection of reversals in protein concentrations or gene expression levels eliminates the need for data normalization or the establishment of population-wide thresholds. Encompassed within the definition of any reversal pair is the corresponding reversal pair wherein individual biomarkers are switched between the numerator and denominator. One skilled in the art will appreciate that such a corresponding reversal pair is equally informative with regard to its predictive power.

The term “reversal value” refers to the ratio of the relative peak areas corresponding to the abundance of two analytes and serves to both normalize variability and amplify diagnostic signal. In some embodiments, a reversal value refers to the ratio of the relative peak area of an up-regulated (interchangeably referred to as “over-abundant,” up-regulation as used herein simply refers to an observation of relative abundance) analyte over the relative peak area of a down-regulated analyte (interchangeably referred to as “under-abundant,” down-regulation as used herein simply refers to an observation of relative abundance). In some embodiments, a reversal value refers to the ratio of the relative peak area of an up-regulated analyte over the relative peak area of a up-regulated analyte, where one analyte differs in the degree of up-regulation relative the other analyte. In some embodiments, a reversal value refers to the ratio of the relative peak area of a down-regulated analyte over the relative peak area of a down-regulated analyte, where one analyte differs in the degree of down-regulation relative the other analyte.

One advantageous aspect of a reversal is the presence of complementary information in the two analytes, so that the combination of the two is more diagnostic of the condition of interest than either one alone. Preferably the combination of the two analytes increases signal-to-noise ratio by compensating for biomedical conditions not of interest, pre-analytic variability and/or analytic variability. Out of all the possible reversals within a narrow window, a subset can be selected based on individual univariate performance. Additionally, a subset can be selected based on bivariate or multivariate performance in a training set, with testing on held-out data or on bootstrap iterations. For example, logistic or linear regression models can be trained, optionally with parameter shrinkage by L1 or L2 or other penalties, and tested in leave-one-out, leave-pair-out or leave-fold-out cross-validation, or in bootstrap sampling with replacement, or in a held-out data set. In some embodiments, the analyte value is itself a ratio of the peak area of the endogenous analyte over that of the peak area of the corresponding stable isotopic standard analyte, referred to herein as: response ratio or relative ratio. As disclosed herein, the ratio of the relative peak areas corresponding to the abundance of two analytes, for example, the ratio of the relative peak area of an up-regulated biomarker over the relative peak area of a down-regulated biomarker, referred herein as a reversal value, can be used to identify robust and accurate classifiers and predict EDD, GAB, and/or predicting time to birth (TTB). Use of a ratio of biomarkers in the methods disclosed herein corrects for variability that is the result of human manipulation after the removal of the biological sample from the pregnant female. Such variability can be introduced, for example, during sample collection, processing, depletion, digestion or any other step of the methods used to measure the biomarkers present in a sample and is independent of how the biomarkers behave in nature. Accordingly, the invention generally encompasses the use of a reversal pair in a method of diagnosis or prognosis to reduce variability and/or amplify, normalize or clarify diagnostic signal.

While the term reversal value refers to the ratio of the relative peak area of an up regulated analyte over the relative peak area of a down regulated analyte and serves to both normalize variability and amplify diagnostic signal, it is also contemplated that a pair of biomarkers of the invention could be measured by any other means, for example, by subtraction, addition or multiplication of relative peak areas. The methods disclosed herein encompass the measurement of biomarker pairs by such other means.

This method is advantageous because it provides the simplest possible classifier that is independent of data normalization, helps to avoid overfitting, and results in a very simple experimental test that is easy to implement in the clinic. The use of marker pairs based on changes in reversal values that are independent of data normalization enabled the development of the clinically relevant biomarkers disclosed herein. Because quantification of any single protein is subject to uncertainties caused by measurement variability, normal fluctuations, and individual related variation in baseline expression, identification of pairs of markers that may be under coordinated, systematic regulation enables robust methods for individualized diagnosis and prognosis.

While the specification discloses embodiments directed to measuring the particular pairs of biomarkers disclosed in Tables 1-27, the invention is not restricted to the particular pairs recited in Tables 1-27 and individual biomarkers disclosed herein as well as any pair or panel of the individual biomarkers is also encompassed by the present invention, as are methods comprising one or more pairs of biomarkers.

In one embodiment, the present invention provides a composition comprising a pair of isolated biomarkers consisting of cathepsin D (CATD) and tenascin X (TENX), wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention provides a pair of surrogate peptides of a pair of biomarkers selected from the group consisting of CATD and TENX, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days. In one embodiment, the present invention further provides stable isotope labeled standard peptides (SIS peptides) corresponding to each of the surrogate peptides.

In one embodiment, the present invention provides a method for separating a pregnancy that delivers before 270 days from a pregnancy that delivers on or after 280 days comprising measuring a change in reversal value of a biomarker pair consisting of CATD and TENX, wherein said pair of biomarkers exhibits a change in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver on or after 280 days.

In one embodiment, the present invention further provides a method for prediction of gestational age at birth (GAB).

In a further embodiment, the present invention further provides a method for prediction of time to birth (TTB).

The present invention further contemplates that the methods and compositions can encompass changes in reversal value between pregnant females that deliver before 270 days relative to pregnant females that deliver, for example, on or after 280 days; before 260 days relative to pregnant females that deliver on or after 270 days; before 250 days relative to pregnant females that deliver on or after 260 days; before 240 days relative to pregnant females that deliver on or after 250 days; before 230 days relative to pregnant females that deliver on or after 240 days. One skilled in the art will be able to select additional time windows, time windows with different cut-offs as well as time windows with different gaps, for example, 5 days, 15 days or 20 days. All of these variations are contemplated by the invention disclosed herein.