Compositions and methods for detecting multiple pregnancy complications

Abstract

This disclosure provides compositions and methods for determining the probability of adverse pregnancy-related outcomes and providing preventive intervention for an adverse pregnancy-related outcome in a pregnant patient. The methods provided herein include measuring in a biological sample obtained from said pregnant patient levels of isolated biomarkers; and calculating a risk score using a reversal value for the levels of isolated biomarkers. The risk score can be used to determine the probability of the adverse pregnancy-related outcome and to determine the need of administration of a preventive intervention for the adverse pregnancy-related outcome.

Description

Attorney Docket No. 203123-030002 / PCTCOMPOSITIONS AND METHODS FOR DETECTING MULTIPLE PREGNANCY COMPLICATIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of United States Provisional Application No. 63 / 754,959, filed February 6, 2025, the entire contents of which are incorporated herein by reference.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on February 5, 2026, is named 203123-030002-PCT_SL.xml and is 42,713 bytes in size.FIELD OF INVENTION

[0003] The disclosure relates generally to the field of precision medicine and, more specifically to compositions and methods for determining the probability of adverse pregnancy-related outcomes in a pregnant patient.BACKGROUND

[0004] Between 2010 and 2020, approximately 13 million infants were born prematurely worldwide. Although targeted proteomic studies have identified a few biomarkers capable of predicting single conditions, such as preterm birth or preeclampsia, there is currently no comprehensive, clinically actionable diagnostic screening or test available that reliably identifies a wide range of adverse pregnancy-related outcomes. These outcomes include preterm birth, preeclampsia, gestational diabetes, gestational hypertension, early (non-elective) cesarian section, extended hospital stays for both mothers and babies, neonatal complications, low birthweight, and respiratory distress syndrome. Such adverse pregnancy-related outcomes are significant risk factors for neonatal death and complications later in life, and can also negatively impact maternal health.

[0005] Despite extensive research efforts to identify at-risk women, existing prediction algorithms for adverse pregnancy-related outcomes, which rely solely on either clinical and demographic factors or using measured biomarkers, have resulted in tests that focus on single conditions. There is a critical need for more accurate methods to identify women at risk of1ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTmultiple adverse pregnancy-related outcomes, especially during their first pregnancy and sufficiently early in gestation to allow for timely clinical intervention. The present disclosure addresses this need by providing compositions and methods for determining whether a pregnant woman is at risk for a plurality of adverse pregnancy-related outcomes. Related advantages are also provided.SUMMARY

[0006] In one aspect of the present disclosure, provided herein is a method of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant human patient. In some embodiments, the method comprises (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 and SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG). In some embodiments, the one or more risk scores are connected to the occurrence of the plurality of adverse pregnancy-related outcomes. In some embodiments, a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, indicates an increased probability of at least one adverse pregnancy-related outcome occurring. In some embodiments, the plurality of adverse pregnancy-related outcomes comprise two or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0007] In some embodiments, the method further comprises combining said reversal value with one or more clinical factors to calculate said one or more risk scores for the plurality of adverse pregnancy-related outcomes occurring, wherein said one or more clinical factors is selected from the group consisting of age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy. In some embodiments, the method further comprises an initial step of2ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTidentifying said pregnant human patient as potentially at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium. In some embodiments, said one or more risk indicium is selected from the group consisting of age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortions, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and / or uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections. In some embodiments, said one or more risk indicium includes BMI. In some embodiments, said BMI is greater than 22 kg / m2 and less than or equal to 37 kg / m2.

[0008] In some embodiments, said biological sample is selected from the group consisting of whole blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine. In one embodiment, said biological sample is whole blood, plasma, or serum. In one embodiment, said biological sample is obtained between 19 and 21 weeks of estimated gestational age.

[0009] In some embodiments of the method of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant human patient, said measuring comprises subjecting the biological sample to a proteomics workflow comprises mass spectrometry (MS) or immunoassay. In one embodiment of the method of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant human patient, said proteomics workflow comprises quantification of a stable isotope labeled (SIS) surrogate peptide of said isolated biomarkers. In one embodiment of the method of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant human patient, said MS is selected from the group consisting of: matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS); matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD)); matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI- TOF / TOF); surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS); electrospray ionization mass spectrometry (ESI-MS); electrospray ionization-mass spectrometry / mass spectrometry (ESI-MS / MS); electrospray ionization mass spectrometry / mass spectrometryn (ESI-MS / (MS)n, wherein n is an integer greater than zero; electrospray ionization 3D (ESI 3D)3ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTor linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS; electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS); electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems); desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCI-MS); atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCI-MS / MS); atmospheric pressure chemical ionizationmass spectrometryn (APCI-(MS)n), wherein n is an integer greater than zero; ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS); atmospheric pressure photoionization mass spectrometry (APPI-MS); atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPI-MS / MS); and atmospheric pressure photoionization-mass spectrometryn (APPI-(MS)n), wherein n is an integer greater than zero. In one embodiment, said MS comprises co-immunoprecipitation-mass spectrometry (co-IPMS). In one embodiment, said MS comprises liquid chromatography-mass spectrometry (LC-MS). In one embodiment, said MS comprises multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

[0010] In some embodiments, the method further comprises communicating said adverse pregnancy-related outcomes to a health care provider. In one embodiment, said communicating informs a subsequent treatment decision for said pregnant human patient and / or a neonate carried by said pregnant human patient. In one embodiment, when the plurality of adverse pregnancy-related outcomes comprises risk for preterm birth and a higher risk score for preterm birth, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of aspirin, a progesterone treatment, tocolytics to delay labor, transvaginal cervical length screenings, weekly nurse telephonic support, bed rest, or reduced physical activity. In one embodiment, said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-ahydroxyprogesterone caproate, or vaginal progesterone. In one embodiment, said 17-a hydroxyprogesterone caproate is administered as an injection. In one embodiment, said vaginal progesterone is administered in gel form. In one embodiment, the preventive intervention further comprises transvaginal cervical length screenings. In some embodiments, when the plurality of adverse pregnancy-related outcomes comprises risk for preeclampsia and a higher risk score for preeclampsia, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of magnesium4ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTsulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity and bed rest.

[0011] In some embodiments, the plurality of adverse pregnancy-related outcomes comprises risk for GAB being less than 37 weeks and a higher risk score for GAB being less than 37 weeks, (i) the treatment decision for the pregnant human patient comprises a preventive intervention comprising administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity, and / or (ii) the treatment decision for the neonate carried by said pregnant human patient comprises a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU). In one embodiment, the plurality of adverse pregnancy-related outcomes comprises risk for GDM and a higher risk score for GDM, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of insulin, an oral hypoglycemic agent, medical nutrition therapy, increased physical activity, and weight management. In one embodiment, when the plurality of adverse pregnancy-related outcomes comprises risk for gHTN and a higher risk score for gHTN, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication, fetal monitoring, hospitalization, and bedrest. In one embodiment, when the plurality of adverse pregnancy-related outcomes comprises risk for low birthweight and a higher risk score for low birthweight, the treatment decision for the neonate comprises a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU). In one embodiment, when the plurality of adverse pregnancy-related outcomes comprises risk for RDS and a higher risk score for RDS, the treatment decision for said neonate comprises a preventive intervention comprising one or more of surfactant therapy, oxygen therapy, continuous airway pressure, mechanical ventilation, nutritional support, and prone positioning.

[0012] In some embodiments, said measuring comprises contacting a capture agent that binds to IBP4 or SHBG to the biological sample. In one embodiment, said capture agent is selected from the group consisting of an antibody, antibody fragment, nucleic acid-based protein binding reagent, small molecule or variant thereof. In one embodiment, said contacting5ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTis part of an assay selected from the group consisting of enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). In one embodiment, said measuring further comprises mass spectrometry (MS). In one embodiment, said MS is coimmunoprecipitation-mass spectrometry (co-IP MS).

[0013] In some embodiments, (a) the isolated biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from the group consisting of QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYIIPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID NO: 4), and / or (b) the isolated biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from the group consisting of IALGGLLFPASNLR (SEQ ID NO: 5), GEDSSTSFCLNGLWAQGQR (SEQ ID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11).

[0014] In another aspect, the present disclosure provides a method of determining the probability of a plurality of adverse pregnancy-related outcomes, wherein the method comprises (i) measuring in a biological sample obtained from a pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating a risk score using a reversal value for IBP4 and SHBG. In one embodiment, the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG). In one embodiment, the risk score is connected to the plurality of adverse pregnancy-related outcomes occurring. In one embodiment, the risk score indicates an increased probability of two or more of the adverse pregnancy-related outcomes when the risk score is at or over 15% compared to a reference risk score, wherein the reference risk score is an algorithm-based biomarker-scored preterm birth risk. In one embodiment, said plurality of adverse pregnancy-related outcomes comprises at least two or more of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0015] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient.6ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTIn some embodiments, the method comprises (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG), wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay; (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) administering to said pregnant human patient, where a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of aspirin, a progesterone treatment, tocolytics to delay labor, transvaginal cervical length screenings, weekly nurse telephonic support, bed rest, and / or reduced physical activity. In one embodiment, the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring. In one embodiment, the plurality of adverse pregnancy-related outcomes comprises preterm birth (PTB) and one or more adverse pregnancy -related outcomes selected from the group consisting of preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0016] In some embodiments, said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-a hydroxyprogesterone caproate, or vaginal progesterone. In one embodiment, said 17-a hydroxyprogesterone caproate is administered as an injection. In one embodiment, said vaginal progesterone is administered in gel form.

[0017] In one aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG), wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay; (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said7ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTSHBG (IBP4 / SHBG); and (iii) administering to said pregnant human patient, where a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of magnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity and / or bed rest. In one embodiment, the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring. In one embodiment, the plurality of adverse pregnancy-related outcomes comprises preeclampsia (PE) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0018] In some embodiments, said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-a hydroxyprogesterone caproate, or vaginal progesterone. In one embodiment, said 17-a hydroxyprogesterone caproate is administered as an injection. In one embodiment, said vaginal progesterone is administered in gel form.

[0019] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising: (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) administering to said pregnant human patient, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of magnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity and / or bed rest, wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, and wherein the plurality of adverse pregnancy -related8ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCToutcomes comprises preeclampsia (PE) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS). In some embodiments, the measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

[0020] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient and / or a neonate carried by said pregnant human patient, the method comprising: (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) where if the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, the method comprises: (a) administering to said pregnant human patient a preventive intervention comprising administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity, and / or (b) administering to the neonate a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, and wherein the plurality of adverse pregnancy-related outcomes comprises predicted gestational age at birth (GAB) being less than 37 weeks and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS). In some embodiments, the measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.9ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0021] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising: (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) administering to said pregnant human patient, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of insulin, an oral hypoglycemic agent, medical nutrition therapy, increased physical activity, and weight management, wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, and wherein the plurality of adverse pregnancy-related outcomes comprises gestational diabetes mellitus (GDM) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS). In some embodiments, the measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

[0022] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising: (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) administering to said pregnant human patient, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication, fetal monitoring, hospitalization, and bedrest, wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,10ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTand wherein the plurality of adverse pregnancy-related outcomes comprises gestational hypertension (gHTN) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS). In some embodiments, the measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

[0023] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient, the method comprising: (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) administering to said neonate, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, and wherein the plurality of adverse pregnancy-related outcomes comprises low birthweight and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and respiratory distress syndrome (RDS). In some embodiments, the measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

[0024] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient, the method comprising: (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like11ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTGrowth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and (iii) administering to said neonate, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of surfactant therapy, oxygen therapy, continuous airway pressure, mechanical ventilation, nutritional support, and prone positioning, wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, and wherein the plurality of adverse pregnancy-related outcomes comprises respiratory distress syndrome (RDS) in the neonate and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and low birthweight. In some embodiments, the measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

[0025] In some embodiments, the methods herein further comprise combining said reversal value with one of more clinical factors selected from the group consisting of age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy to calculate said one or more risk scores for the plurality of adverse pregnancy-related outcomes occurring. In one embodiment, the methods further comprise an initial step of identifying said pregnant human patient as potentially at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium. In one embodiment, said one or more risk indicium is selected from the group consisting of age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections. In one embodiment, said one or more12ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTrisk indicium includes BMI. In one embodiment, said BMI is greater than 22 kg / m2 and less than or equal to 37 kg / m2. In one embodiment, said biological sample is selected from the group consisting of whole blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine. In one embodiment, said biological sample is whole blood, plasma or serum. In one embodiment, said biological sample is obtained between 19 and 21 weeks of estimated gestational age.

[0026] In some embodiments of the methods herein, said proteomics workflow comprises quantification of a stable isotope labeled (SIS) surrogate peptide of said isolated biomarkers. In one embodiment, said MS is selected from the group consisting of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS); matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD)); matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI-TOF / TOF); surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS); electrospray ionization mass spectrometry (ESI-MS); electrospray ionization-mass spectrometry / mass spectrometry (ESLMS / MS); electrospray ionization mass spectrometry / mass spectrometryn (ESI-MS / (MS)n, wherein n is an integer greater than zero; electrospray ionization 3D (ESI 3D) or linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS; electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS); electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems); desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCLMS); atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCI-MS / MS); atmospheric pressure chemical ionization-mass spectrometryn (APCL(MS)n), wherein n is an integer greater than zero; ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS); atmospheric pressure photoionization mass spectrometry (APPLMS); atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPLMS / MS); and atmospheric pressure photoionization-mass spectrometryn (APPL (MS)n), wherein n is an integer greater than zero. In one embodiment, said MS comprises coimmunoprecipitation-mass spectrometry (co-IP MS). In one embodiment, said MS comprises liquid chromatography-mass spectrometry (LC-MS). In one embodiment, said MS comprises multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).13ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0027] In some embodiments of the methods herein, (a) the isolated biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from the group consisting of QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYIIPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID NO: 4), and / or (b) the isolated biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from the group consisting of IALGGLLFPASNLR (SEQ ID NO: 5), GEDSSTSFCLNGLWAQGQR (SEQ ID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11).

[0028] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of aspirin, a progesterone treatment, tocolytics to delay labor, transvaginal cervical length screenings, weekly nurse telephonic support, bed rest, and / or reduced physical activity. In one embodiment, said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores. In one embodiment, the plurality of adverse pregnancy-related outcomes comprises preterm birth (PTB) and one or more adverse pregnancy-related outcomes selected from the group consisting of preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0029] In some embodiments, said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-a hydroxyprogesterone14ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTcaproate, or vaginal progesterone. In one embodiment, said 17-a hydroxyprogesterone caproate is administered as an injection. In one embodiment, said vaginal progesterone is administered in gel form.

[0030] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of magnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity, and bed rest, wherein said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and wherein the plurality of adverse pregnancy-related outcomes comprises preeclampsia (PE) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0031] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient and / or a neonate carried by said pregnant human patient by (a) administering to said pregnant human patient a preventive intervention comprising administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity, and / or (b) administering to said neonate a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU), wherein said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said15ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTpregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and wherein the plurality of adverse pregnancy-related outcomes comprises predicted gestational age at birth (GAB) being less than 37 weeks and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0032] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of insulin, an oral hypoglycemic agent, medical nutrition therapy, increased physical activity, and weight management, wherein said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and wherein the plurality of adverse pregnancy-related outcomes comprises gestational diabetes mellitus (GDM) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).16ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0033] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication, fetal monitoring, hospitalization, and bedrest, wherein said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and wherein the plurality of adverse pregnancy-related outcomes comprises gestational hypertension (gHTN) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

[0034] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient by administering to said neonate a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, or a stay in a neonatal intensive care unit (NICU), wherein said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy -related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference17ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTrisk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and wherein the plurality of adverse pregnancy-related outcomes comprises low birthweight and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and respiratory distress syndrome (RDS).

[0035] In another aspect, the present disclosure provides a method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient by administering to said neonate a preventive intervention comprising one or more of surfactant therapy, oxygen therapy, continuous airway pressure, mechanical ventilation, nutritional support, or prone positioning, wherein said pregnant human patient has been identified for treatment by (i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and (ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG), wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and wherein the plurality of adverse pregnancy-related outcomes comprises respiratory distress syndrome (RDS) in the neonate and one or more adverse pregnancy -related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and low birthweight.

[0036] In some embodiments of the methods herein, (a) the isolated biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from the group consisting of QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYI IPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID NO: 4), and / or (b) the isolated biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from the group consisting of IALGGLLFPASNLR (SEQ ID NO:18ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT5), GEDSSTSFCLNGLWAQGQR (SEQ ID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11). In one embodiment, any one of the above methods further comprise combining said reversal value with one of more clinical factors selected from the group consisting of age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy to calculate said one or more risk scores for the plurality of adverse pregnancy-related outcomes occurring. In some embodiments, the methods further comprise an initial step of identifying said pregnant human patient as potentially at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium. In one embodiment, said one or more risk indicium is selected from the group consisting of age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections. In one embodiment, wherein said one or more risk indicium includes BMI. In one embodiment, said BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2. In one embodiment, said biological sample is selected from the group consisting of whole blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine. In one embodiment, said biological sample is whole blood, plasma or serum. In one embodiment, said biological sample is obtained between 19 and 21 weeks of estimated gestational age.

[0037] In some embodiments, measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay. In one embodiment, said proteomics workflow comprises quantification of a stable isotope labeled (SIS) surrogate peptide of said biomarkers. In one embodiment, said MS is selected from the group consisting of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS); matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD)); matrix-assisted laser19ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTdesorption / ionization time-of-flight / time-of-flight (MALDI- TOF / TOF); surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS); electrospray ionization mass spectrometry (ESI-MS); electrospray ionization-mass spectrometry / mass spectrometry (ESI-MS / MS); electrospray ionization mass spectrometry / mass spectrometryn (ESI-MS / (MS)n, wherein n is an integer greater than zero; electrospray ionization 3D (ESI 3D) or linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS; electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS); electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems); desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCI-MS); atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCI-MS / MS); atmospheric pressure chemical ionizationmass spectrometryn (APCI-(MS)n), wherein n is an integer greater than zero; ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS); atmospheric pressure photoionization mass spectrometry (APPI-MS); atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPI-MS / MS); and atmospheric pressure photoionization-mass spectrometryn (APPI-(MS)n), wherein n is an integer greater than zero. In one embodiment, said MS comprises co-immunoprecipitation-mass spectrometry (co-IPMS). In one embodiment, said MS comprises liquid chromatography-mass spectrometry (LC-MS). In one embodiment, said MS comprises multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 provides a plot showing the performance of a predictor of the present disclosure, where “sens” indicates sensitivity, “PPV” indicates positive predictive value, and “NPV” indicates negative predictive value. The y-axis of the plot of FIG. 1 represents the fraction of patients having an adverse pregnancy outcome. In FIG. 1, “(assay)” indicates a predictor incorporating clinical and demographic factors as well as reversal value scores (i.e., a predictor based on a “combined score”), and the absence of the designator “(assay)” indicates a predictor using clinical and demographic factors alone and no reversal value scores.

[0039] FIG. 2 provides a plot showing the performance of a predictor of the present disclosure, where “High NNOLOS” indicates a long hospital stay for the baby (i.e., neonatal length of stay in NICU), “High MLOS” indicates a long hospital stay for the mother (i.e., long20ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTmaternal length of stay), and “C-section” indicates an unplanned C-section. The y-axis of the plot of FIG. 2 represents the number of occurrences of each indicated adverse pregnancy outcome detected using the predictor. In FIG. 2, “(assay)” indicates a predictor incorporating clinical and demographic factors as well as reversal value scores (i.e., a predictor based on a “combined score”), and “(risk factors)” indicates a predictor using clinical and demographic factors alone and no reversal value scores.DETAILED DESCRIPTION

[0040] Aspects of the present disclosure relate to methods of determining the probability of adverse pregnancy-related outcomes in a pregnant patient, and methods for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant patient. Such methods may include detecting a condition (e.g., placental dysfunction) that gives rise to an increased probability of a plurality of adverse pregnancy outcomes that result from such condition (e.g., preterm birth, preeclampsia, gestational diabetes, gestational hypertension, early (non-elective) cesarean section, extended hospital stays, neonatal complications, low birthweight, and respiratory distress syndrome).

[0041] The methods disclosed herein are based, generally, on the discovery that certain proteins and peptides in biological samples obtained from a pregnant human patient are differentially expressed in pregnant human patients that have an increased risk of a plurality of adverse pregnancy-related outcomes relative to controls. The methods disclosed herein are further specifically based, in part, on the unexpected discovery that reversal values of pairs of biomarkers disclosed herein can be utilized in methods of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant human patient with high sensitivity and specificity. The proteins and peptides disclosed herein as components of ratios and / or reversal pairs serve as biomarkers for classifying test samples, predicting probability of a plurality of adverse pregnancy-related outcomes, either individually, in ratios, reversal pairs or in panels of biomarkers / reversal pairs. A reversal value is typically the ratio of the level (e.g., relative peak area) of an upregulated biomarker over the level (e.g., relative peak area) of a downregulated biomarker (or vice versa) in a sample and serves to both normalize variability and amplify diagnostic signal. The methods disclosed herein rely, in part, in the selection of particular biomarkers that, when paired together, can predict the probability of a plurality of adverse pregnancy -related outcomes based on reversal values. In other words, the methods and compositions disclosed herein utilize specific pairs of biomarkers, where the ratio of an21ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTupregulated biomarker to a downregulated biomarker (reversal value) is used to predict adverse pregnancy-related outcomes. Accordingly, it is human ingenuity, inter alia, in selecting the specific biomarkers that are informative upon being paired in novel reversals that underlies the present disclosure.

[0042] While the term reversal value typically refers to the ratio of the level of an upregulated analyte over the level of a downregulated analyte (or vice versa) and serves to both normalize variability and amplify diagnostic signal, it is also contemplated that a pair of biomarkers of the disclosure could be measured by a variety means, for example, by relative peak area, including subtraction, addition or multiplication of relative peak areas. The methods disclosed herein encompass the measurement of biomarker pairs by such means.

[0043] This technique of using reversal values is advantageous because it provides a simple 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.

[0044] The disclosure provides biomarker reversal pairs and associated panels of reversal pairs, methods and kits for determining the probability for a plurality of adverse pregnancy-related outcomes in a pregnant human patient. One major advantage of the present disclosure is that risk of developing a plurality of adverse pregnancy-related outcome can be assessed early during pregnancy so that appropriate monitoring and clinical management to prevent one or more of those outcomes can be initiated in a timely fashion. The present disclosure is of particular benefit to human patients lacking any traditional risk factors for the adverse pregnancy-related outcome and who would not otherwise be identified and treated. Moreover, unlike existing methods that focus on predicting a single outcome, the methods of the present disclosure can predict a wide range of adverse pregnancy-related outcomes, including preterm birth, preeclampsia, gestational diabetes, gestational hypertension, early (non-elective) cesarean section, extended hospital stays, neonatal complications, low birthweight, and respiratory distress syndrome.22ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0045] By way of example, the present disclosure includes methods for generating a result useful in determining probability for a plurality of adverse pregnancy-related outcomes in a pregnant human patient by obtaining a dataset associated with a sample, where the dataset at least includes quantitative data about the relative amounts of biomarker pairs that have been identified as exhibiting changes in reversal value predictive of adverse pregnancy-related outcomes, and inputting the dataset into an analytic process that uses the dataset to generate a result useful in determining probability for adverse pregnancy-related outcomes in a pregnant human patient. As described further below, quantitative data can include the amounts or levels of amino acids, peptides, polypeptides, proteins, nucleotides, nucleic acids, nucleosides, sugars, fatty acids, steroids, metabolites, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as surrogates for biological macromolecules and combinations thereof.

[0046] In addition to the specific biomarkers identified in this disclosure, for example, by accession number in a public database, sequence, or reference, the disclosure 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 disclosure. These variants may represent polymorphisms, splice variants, mutations, and the like. As described herein, various techniques and reagents find use in the methods of the present disclosure. Suitable samples in the context of the present disclosure 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.

[0047] The methods described herein can also combine biomarker data with clinical factors such as age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy, to improve predictive accuracy. This integrated approach enhances the ability to identify at-risk pregnancies more accurately. The compositions and methods described herein can not only be used to predict23ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTadverse outcomes (e.g., detect the present condition(s) that give rise to an increased risk of such outcomes), but also provide methods for administering preventive interventions based on such risk. This proactive approach can significantly improve maternal and neonatal health outcomes. The compositions and methods can also generate personalized risk scores based on the biomarker reversal values (and the presence of the condition to be detected), providing a tailored assessment of the likelihood of adverse pregnancy-related outcomes for each individual patient.

[0048] Overall, the compositions and methods described herein represent a significant advancement in the field of precision medicine for pregnancy management, offering a comprehensive, accurate, and early detection strategy for a wide range of adverse pregnancy-related outcomes, along with actionable insights for preventive intervention.I. Definitions

[0049] The following definitions supplement those in the art and are directed to the present disclosure only. The following definitions are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or patent application. Although some methods and materials similar or equivalent to those described herein can be used to practice features of the disclosure, some exemplary materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0050] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0051] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0052] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.24ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0053] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0054] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some instances, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0055] 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.

[0056] As used herein, the terms “administration,” “administering” and variants thereof refer to the introduction of a therapeutic agent into a subject or performance of a therapeutic action on a subject. Administration of a composition or therapeutic agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. A suitable route of administration allows the composition or the agent to perform its intended function. Administration also includes self-administration and the administration by another. The administration can also be performed systemic, or it can be local.

[0057] As used herein, the term “adverse pregnancy-related outcome” refers to any negative health event or complication during pregnancy, childbirth, or the postpartum period, including but not limited to preterm birth, preeclampsia, gestational diabetes, gestational hypertension, early (non-elective) cesarean section, extended hospital stays, neonatal complications, low birthweight, and respiratory distress syndrome in the neonate. In some instances, the adverse pregnancy-related outcome can affect the mother (e.g., pregnant female), the baby (including a fetus or neonate), or both. In some instances, low birthweight refers to a25ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTneonate who weighs less than 5 pounds, 8 ounces (2,500 grams) at birth. In some instances, the adverse pregnancy-related outcome can include a disease or condition associated with pregnancy. In some instances, the adverse pregnancy-related outcome is an event associated with still birth, miscarriage, neonatal death and / or complications later in life for the mother or baby, such as extended hospital stays and neonatal complications. In some instances, a negative health event includes an unfavorable or sub-optimal event.

[0058] As used herein, the term “biological sample” refers to any sample that is taken from a pregnant human patient and contains one or more of the biomarkers disclosed herein. Suitable samples in the context of the present disclosure include, for example, blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine. In some instances, the biological sample is selected from the group consisting of whole blood, plasma, and serum. In some instances, the biological sample is selected from the group consisting of dried whole blood, dried plasma, and dried serum. In a particular instance, the biological sample is serum. As will be appreciated by those skilled in the art, a biological sample can include any fraction or component of blood, without limitation, T cells, monocytes, neutrophils, erythrocytes, platelets and micro-vesicles such as exosomes and exosome-like vesicles.

[0059] As used herein, the term “biomarker” refers to a biological molecule, or a fragment of a biological molecule, the state, structure, sequence, amount, level, change and / or the detection of which can be correlated with a particular physical condition or state. Except where expressly stated or context clearly indicates otherwise, the terms “marker” and “biomarker” are used interchangeably throughout the disclosure. For example, the biomarkers of the present disclosure are correlated with an increased likelihood of a plurality of adverse pregnancy-related outcomes (e.g., outcomes caused by or contributed to by pregnancy conditions, such as placental dysfunction). 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 1026ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTconsecutive 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 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.

[0060] As used herein, the term “clinical factor” refers to a patient-specific factor (e.g., age, BMI, medical history, and other relevant health indicators) used to improve predictive accuracy, including but not limited to race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, C-section delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy. In some instances, a clinical factor is used in the methods of the disclosure (e.g., when combined with biomarkers).

[0061] As used herein, the term “estimated gestational age” or “estimated GA” refers to the GA determined based on the first day of the last normal menstrual period (LMP) and / or additional obstetric measures, ultrasound estimates or other clinical parameters including, without limitation, those described in the preceding paragraph. Establishing an estimated GA also allows the determination of an estimated due date (EDD), taken by Naegale’s rule as EDD = first day of LMP + 280 days (40 weeks). In contrast, the term “predicted gestational age at birth” or “predicted GAB” refers to the GAB determined based on the methods of the embodiments as disclosed herein, including a model-derived prognostic value that estimates, in units of weeks and / or days, the gestational-age - equivalent developmental maturity anticipated at delivery based on one or more biomarker reversal values and, optionally, clinical and demographic variables. Predicted GAB may correct for the fetus’s developmental trajectory rather than a measure of elapsed time in utero. Because fetal development rates can vary among pregnancies, the predicted GAB may differ from the obstetric dating of pregnancy length and does not purport to quantify the actual amount of time the fetus has been carried. Predicted GAB, may also correct for inaccuracies in the estimated GA, due to deviations in assumptions made for Naegale’s rule (e.g. inaccurate recollection of the last menstrual period,27ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTdeviations from a normal 28-day cycle or ovulation not occurring on day 14), or accurate determination of fetal size by ultrasound. Likewise, predicted GAB may correct for adverse pregnancy conditions or states, that shorten or lengthen the gestational period. Examples here include placental insufficiency, infection and other chronic or acute medical conditions. Predicted GAB can include GABs prior to 37 weeks, but is also distinct from “preterm birth,” which is an outcome defined by actual delivery before 37 completed weeks of gestation by obstetric dating. Accordingly, references to “predicted GAB being less than 37 weeks” indicate a likelihood classification that the fetus is predicted to reach a developmental state corresponding to, or an adverse pregnancy condition associated with, delivery before 37 completed weeks and is used to stratify likelihood and guide management; it neither requires nor implies that a preterm birth has occurred. As used herein, “term birth” refers to birth at a gestational age between weeks 37 and 41 of gestation (inclusive).

[0062] As used herein, the terms “individual,” “patient” and “subject” interchangeably refer to any animal, including, but not limited to, humans, non-human primates, rodents, and domestic and game animals, which is to be the recipient of a particular treatment. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. In some instances, a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment. In some instances, the subject is a human. In some instances, the subject previously diagnosed with or identified as suffering from or having a condition may or may not have undergone treatment for a condition. In yet other instances, a subject can also be one who has not been previously diagnosed as having a condition (i.e., a subject who exhibits one or more risk factors for a condition). A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0063] 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 at28ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTleast 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.

[0064] As used herein, the terms “mass spectrometry” or “MS” refer to an analytical technique used to measure the mass-to-charge ratio of ions, useful for identifying and quantifying molecules in a sample. Such a technique is performed using a mass spectrometer. Mass spectrometry can also encompass additional techniques as described herein and be included as part of a proteomics workflow.

[0065] As used herein, the term “mass spectrometer” refers to a device able to volatilize / ionize analytes to form gas-phase ions and determine their absolute or relative molecular masses. Suitable methods of volatilization / ionization are matrix-assisted laser desorption ionization (MALDI), electrospray, laser / light, thermal, electrical, atomized / sprayed and the like, or combinations thereof. Suitable forms of mass spectrometry include, but are not limited to, ion trap instruments, quadrupole instruments, electrostatic and magnetic sector instruments, time of flight instruments, time of flight tandem mass spectrometer (TOF MS / MS), Fourier-transform mass spectrometers, Orbitraps and hybrid instruments composed of various combinations of these types of mass analyzers. These instruments can, in turn, be interfaced with a variety of other instruments that fractionate the samples (for example, liquid chromatography or solid-phase adsorption techniques based on chemical, or biological properties) and that ionize the samples for introduction into the mass spectrometer, including matrix-assisted laser desorption (MALDI), electrospray, or nanospray ionization (ESI) or combinations thereof.

[0066] As used herein, the terms “multiple reaction monitoring (MRM)” or “selected reaction monitoring (SRM)” refer to an MS-based quantification method that is particularly useful for quantifying analytes that are in low abundance. In an SRM experiment, a predefined precursor ion and one or more of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. Multiple SRM precursor and fragment ion pairs can be measured within the same experiment on the chromatographic time scale by rapidly toggling between the different precursor / fragment pairs to perform an MRM experiment. A series of transitions (precursor / fragment ion pairs) in combination with the retention time of the targeted analyte (e.g., peptide or small molecule such as chemical entity, steroid, hormone) can constitute a definitive assay. A large number of analytes can be quantified during a single LC-MS experiment. The term “scheduled,” or29ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT“dynamic” in reference to MRM or SRM, refers to a variation of the assay wherein the transitions for a particular analyte are only acquired in a time window around the expected retention time, significantly increasing the number of analytes that can be detected and quantified in a single LC-MS experiment and contributing to the selectivity of the test, as retention time is a property dependent on the physical nature of the analyte. A single analyte can also be monitored with more than one transition. Finally, included in the assay can be standards that correspond to the analytes of interest (e.g., same amino acid sequence), but differ by the inclusion of stable isotopes. Stable isotopic standards (SIS) can be incorporated into the assay at precise levels and used to quantify the corresponding unknown analyte. An additional level of specificity is contributed by the co-elution of the unknown analyte and its corresponding SIS and properties of their transitions (e.g., the similarity in the ratio of the level of two transitions of the unknown and the ratio of the two transitions of its corresponding SIS).

[0067] As used herein, the term “preterm birth” refers to delivery or birth before 37 completed weeks of gestation, which may be referred to as 37 weeks 0 days, or 37 0 / 7, or 37w0d. Gestational age is typically counted as full weeks, with 0 through 6 days of gestation between weeks. The 7thday of 7 represents start of the next week, i. e. , day 0 / 7 of the next week. The first day of the last menstrual period (LMP) is week 0 day 0, or 0 0 / 7. Then the next day is 0 weeks 1 day, etc. Thus, 0 weeks and 7 / 7 days is referred to as 1 week 0 / 7 days. Preterm birth is defined as any delivery before 37 weeks 0 days (i.e., before 37 completed weeks) of pregnancy. Estimated due date is equal to 40 0 / 7 or 40 completed weeks of gestation. Unless otherwise indicated, birth at a particular number of weeks of gestation refers to birth after that number of completed weeks of gestation — e.g., birth at 37 weeks of gestation means birth within 37 weeks 0 days and 37 weeks 6 days of gestation. Other commonly used subcategories of preterm birth have been established and delineate moderately preterm (birth at 33 to 36 weeks of gestation, inclusive), very preterm (birth at <33 and >28 weeks of gestation), and extremely preterm (birth at <28 weeks of gestation). With regard to the methods disclosed herein, those skilled in the art understand that the cut-offs that delineate preterm birth and term birth as well as the cut-offs that delineate subcategories of preterm birth can be adjusted in practicing the methods disclosed herein, for example, to maximize a particular health benefit. It is further understood that such adjustments are well within the skill set of individuals considered skilled in the art and encompassed within the scope of the various aspects and embodiments disclosed herein. In various embodiments of the disclosure, the cut-off that delineates preterm birth from term birth includes, for example, birth before 37 weeks of30ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTgestation, before 36 weeks of gestation, before 35 weeks of gestation, before 34 weeks of gestation, before 33 weeks of gestation, before 32 weeks of gestation, before 30 weeks of gestation, before 29 weeks of gestation, before 28 weeks of gestation, before 27 weeks of gestation, before 26 weeks of gestation, before 25 weeks of gestation, before 24 weeks of gestation, before 23 weeks of gestation or before 22 weeks of gestation. In some embodiments, the cut-off delineating preterm birth is before 35 weeks of gestation. As used herein, the phrase “term” refers to birth at or after 37 completed weeks of gestation (e.g., at or after 37 weeks 0 days). Gestational age is generally used in medicine as a proxy for the extent of fetal development and the fetus’s readiness for birth. Gestational age has typically been defined as the length of time from the date of the last normal menses. However, obstetric measures and ultrasound estimates also can aid in determining gestational age. Preterm births have generally been classified into two separate subgroups. One, spontaneous preterm births are those occurring subsequent to spontaneous onset of preterm labor or preterm premature rupture of membranes regardless of subsequent labor augmentation or cesarean delivery. Two, medically-indicated preterm births are those occurring following induction or cesarean section (C-section) for one or more conditions that the woman’s caregiver determines to threaten the health or life of the mother and / or fetus. In some embodiments, the methods disclosed herein are directed to determining the risk or probability for spontaneous preterm birth or medically-indicated preterm birth. In some embodiments, the methods disclosed herein are directed to determining the risk or probability for spontaneous preterm birth. In additional embodiments, the methods disclosed herein are directed to determining the risk or probability for medically indicated preterm birth. In additional embodiments, the methods disclosed herein are directed to predicting gestational age at birth.

[0068] As used herein, the term “preventive intervention” refers to a therapeutic treatment or other activity designed to prevent or reduce the likelihood or incidence of one or more adverse pregnancy-related outcomes in a patient or population of patients, including but not limited to administration of medications, lifestyle modifications, and clinical monitoring. Examples of such preventive interventions are described herein and typically depend upon the adverse pregnancy-related outcome. For example, for preterm birth, preventive intervention can include one or more of administration of progesterone (e.g., progestogen, 17-a hydroxyprogesterone caproate, vaginal progesterone), administration of low-dose aspirin, weekly nurse telephonic support, transvaginal cervical length screenings, bed rest, and / or reduced physical activity, or use of tocolytics to delay labor; and for preeclampsia, preventive31ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTintervention can include one or more of administration of magnesium sulfate, administration of low-dose aspirin, induction of labor, hospitalization and close monitoring, reduced physical activity, bed rest, and / or antihypertensive medications.

[0069] As used herein, the term “proteomics workflow” refers to a series of steps involving the preparation, separation, detection, and / or quantification of proteins and peptides in a biological sample, often involving mass spectrometry. A proteomics workflow may generally encompass one or more of the following steps: Serum samples are thawed and, optionally, depleted of high abundance proteins (e.g, the 14 highest abundance proteins), e.g., by immune-affinity chromatography. Serum (optionally depleted) is digested with a protease, for example, trypsin, to yield peptides. The digest is subsequently fortified with a mixture of SIS peptides and then desalted and subjected to LC-MS / MS with a triple quadrupole instrument operated in MRM mode. Response ratios are formed from the area ratios of endogenous peptide peaks and the corresponding SIS peptide counterpart peaks. Those skilled in the art appreciate that other types of MS such as, for example, MALDI-TOF, or ESI-TOF, can be used in the methods of the disclosure. In addition, one skilled in the art can modify a proteomics workflow, for example, by selecting particular reagents (such as proteases) or omitting or changing the order of certain steps, for example, it may not be necessary to immunodeplete, the SIS peptide could be added earlier or later and stable isotope labeled proteins could be used as standards instead of peptides.

[0070] As used herein, the term “reference score” or “standard score” refers to a predetermined score corresponding to the risk of a condition or outcome representative of (e.g., average, median, standard deviation) a reference cohort of pregnant females (e.g., as derived from the amount of biomarkers detected in), used as a comparison against the risk score of an individual patient (e.g., test score) for detecting or determining the probability or risk for such condition or outcome in such test patient. A reference score can be a standard (e.g., a number) or a threshold (e.g., a line on a graph).

[0071] 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 can eliminate the need for data normalization or the establishment of population-wide thresholds. In some instances, the reversal pair is a pair of isolated biomarkers, wherein the reversal pair exhibits a change in reversal value between pregnant females at risk for a plurality of adverse pregnancy-related outcomes (e.g, wherein a32ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTspecific condition was detected) compared to controls. In a further instance, the reversal pair exhibits a higher ratio in pregnant females at risk for a plurality of adverse pregnancy-related outcomes compared to controls. 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.

[0072] As used herein, the terms “reversal value” or “reversal” refers to a ratio of the level of one biomarker to the level of another (e.g., of an upregulated biomarker to a downregulated biomarker) in a sample (or vice versa), used to normalize variability and amplify diagnostic signals. The reversal value can be calculated using the relative peak area of biomarker in the sample, such that the ratio of the relative peak area of one analyte (e.g, upregulated) over the relative peak area of another analyte (e.g, downregulated, or vice versa) represents the reversal value. Out of all the possible reversals within a narrow window, a subset can be selected based on individual univariate performance. As disclosed herein, the ratio of the relative peak area of one biomarker (e.g., upregulated) over the relative peak area of another (e.g., downregulated biomarker, or vice versa) can be used to identify robust and accurate classifiers and predict probability of a plurality of adverse pregnancy -related outcomes, predicting time to birth and / or monitoring of progress of preventive therapy in a pregnant female.

[0073] As used herein, the term “risk indicia” refers to a factor that is associated with an increased likelihood of an adverse pregnancy-related outcomes in a patient. The risk indicia can be related to maternal characteristics, medical history, past pregnancy history, or obstetrical history. Exemplary risk indicia include, but are not limited to, age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections.

[0074] As used herein, the term “risk score” or “test score” refers to a score (e.g., a numerical score) that can be assigned based on the amount of one or more biomarkers or reversal values in a biological sample obtained from an individual (e.g., a pregnant female).33ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTRisk or test scores can be compared to the amount of the one or more biomarkers calculated from biological samples obtained from a reference group of individuals (e.g., pregnant females) (e.g, a “standard score” or “reference score” that represents a standard or reference (e.g., average, median, standard deviation) amount of the one or more biomarkers detected in biological samples obtained from the reference group of individuals). In some embodiments, the risk score is expressed as the log of the reversal value, i.e., the ratio of the relative intensities of the individual biomarkers. One skilled in the art will appreciate that a risk score can be expressed based on various data transformations as well as being expressed as the ratio itself. Furthermore, with particular regard to reversal pairs, one skilled in the art will appreciate that any ratio is equally informative if the biomarkers in the numerator and denominator are switched or if related data transformations (e.g. subtraction) are applied. Because the level of a biomarker may not be static throughout pregnancy, a standard or reference score generally is in various embodiments obtained from the reference individual (e.g., pregnant female) for the gestational time point that corresponds to that of the test individual at the time her sample was taken. The standard or reference score can be predetermined and built into a predictor model such that the comparison is indirect rather than performed every time the test score or probability is determined for an individual. The value of the risk score correlates to the deviation, upwards or downwards, from the reference score. In certain embodiments, if a risk score or test score is greater than a standard or reference score, the pregnant female can have an increased likelihood of adverse pregnancy-related outcomes. In some embodiments, the magnitude of a pregnant female’s risk score, or the amount by which it exceeds a reference risk score, can be indicative of or correlated to that pregnant female’s quantitative likelihood of adverse pregnancy-related outcomes.

[0075] As used herein, the term “stable isotope labeled (SIS) peptide” refers to a synthetic peptide labeled with stable isotopes, which can be used as an internal standard in mass spectrometry assays to quantify corresponding endogenous peptides.

[0076] 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 other34ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTamino 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.

[0077] As used herein, the terms “treat,” “treating,” and “treatment” as used herein refers to alleviating or abrogating, or attempting to alleviate or abrogate a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating a cause of the disorder, disease, or condition itself. Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis.II. Methods

[0078] Provided herein are methods of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant patient (e.g., by detecting a condition that causes or contributes to such outcomes) and methods for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant patient.

[0079] In some embodiments, the method for determining the probability of adverse pregnancy-related outcomes in a pregnant patient includes (i) measuring in a biological sample obtained from said pregnant patient levels of isolated biomarkers; and (ii) calculating one or more risk scores using a reversal value for the isolated biomarkers. In some embodiments, the one or more risk scores are associated with the plurality of adverse pregnancy-related outcomes occurring. In some embodiments, a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, indicates an increased probability of at least one adverse pregnancy-related outcome occurring.

[0080] In some embodiments, the reversal value is calculated based on the ratio of two biomarkers. In some embodiments, the ratio may include an upregulated protein in the numerator, a downregulated protein in the denominator, or vice versa. For example, as exemplified herein, IBP4 / SHBG is a ratio of an upregulated protein in the numerator and a downregulated protein in the denominator, which is defined herein as a “reversal.” In instances where the ratio includes an upregulated protein in the numerator, or a downregulated protein35ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTin the denominator, the un-regulated protein would serve to normalize (e.g., decrease pre-analytical or analytical variability). In the particular case of a ratio that is a “reversal” both amplification and normalization are possible. It is understood, that the methods of the disclosure are not limited to the subset of reversals, but also encompass ratios of biomarkers.

[0081] This reversal method is advantageous because it provides a simple classifier that can be 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 biomarker pairs based on reversals that are independent of data normalization as described herein has tremendous power as a method for the identification of clinically relevant biomarkers for predicting adverse pregnancy-related outcomes. 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 can be under coordinated, systematic regulation can prove to be more robust for individualized diagnosis and prognosis.

[0082] In some embodiments, the method for determining the probability of adverse pregnancy-related outcomes in a pregnant patient further includes combining the reversal value with one or more clinical factors to determine the plurality of adverse pregnancy-related outcomes in said pregnant human patient. Exemplary clinical factors that can be combined with the reversal value include, but are not limited to, blood draw period, fetal gender, gravidity, age, BMI, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, C-section delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy, as well as any other knowable patient features.

[0083] In some embodiments, the method for determining the probability of adverse pregnancy-related outcomes in a pregnant patient further includes an initial step of identifying said pregnant human patient as at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicia. Exemplary risk indicia include, but are not limited to, maternal characteristics, medical history, past pregnancy history, and obstetrical history. Such additional markers can include, for example, previous low birth weight or preterm delivery, multiple 2ndtrimester spontaneous abortions, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, short cervical length measurements, gestational bleeding, intrauterine36ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTgrowth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy weight, low or high body mass index, diabetes, hypertension, urogenital infections (e.g., urinary tract infection), asthma, anxiety and depression, asthma, hypertension, hypothyroidism. 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 risk indicia 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 RE, Butler AS, editors. Washington (DC): National Academies Press (US); 2007). Additional risk indicia 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. In some embodiments, the one or more risk indicium includes age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections, or any combination thereof.

[0084] In some embodiments, the risk indicium includes BMI. As described herein, the predictive performance of the claimed methods can be improved with a BMI stratification of greater than 22 kg / m2and equal or less than 37 kg / m2. Accordingly, in some embodiments, the methods of the disclosure can be practiced with samples obtained from pregnant human patients with a specified BMI. Briefly, BMI is an individual’s weight in kilograms divided by the square of height in meters. BMI does not measure body fat directly, but research has shown that BMI is correlated with more direct measures of body fat obtained from skinfold thickness measurements, bioelectrical impedance, densitometry (underwater weighing), dual energy x-ray absorptiometry (DXA) and other methods. Furthermore, BMI appears to be as strongly37ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTcorrelated with various metabolic and disease outcome as are these more direct measures of body fatness. Generally, an individual with a BMI below 18.5 is considered underweight, an individual with a BMI of equal or greater than 18.5 to 24.9 normal weight, while an individual with a BMI of equal or greater than 25.0 to 29.9 is considered overweight and an individual with a BMI of equal or greater than 30.0 is considered obese. In some embodiments, the predictive performance of the claimed methods can be improved with a BMI stratification of equal or greater than 18, equal or greater than 19, equal or greater than 20, equal or greater than 21, equal or greater than 22, equal or greater than 23, equal or greater than 24, equal or greater than 25, equal or greater than 26, equal or greater than 27, equal or greater than 28, equal or greater than 29 or equal or greater than 30. In other embodiments, the predictive performance of the claimed methods can be improved with a BMI stratification of equal or less than 18, equal or less than 19, equal or less than 20, equal or less than 21, equal or less than 22, equal or less than 23, equal or less than 24, equal or less than 25, equal or less than 26, equal or less than 27, equal or less than 28, equal or less than 29 or equal or less than 30. In some embodiments, the BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2.

[0085] In some embodiments, the method for determining the probability of adverse pregnancy-related outcomes in a pregnant patient further includes communicating said adverse pregnancy-related outcomes to a health care provider. In some embodiments, said communicating informs a subsequent treatment decision for said pregnant human patient. In some embodiments, the treatment decision for said pregnant human patient comprises a preventive intervention.

[0086] In some embodiments, the method for determining the probability of a plurality of adverse pregnancy-related outcomes includes (i) measuring in a biological sample obtained from said pregnant patient levels of isolated biomarkers; and (ii) calculating one or more risk scores using a reversal value for the isolated biomarkers. In some embodiments, the one or more risk scores are connected to the plurality of adverse pregnancy-related outcomes occurring. In some embodiments, a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, indicates an increased probability of at least one adverse pregnancy-related outcome occurring.

[0087] In some embodiments, the one or more risk scores indicate an increased probability of two or more of the adverse pregnancy-related outcomes when the one or more risk scores38ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTare at or over one or more reference risk scores. In some embodiments, the one or more risk scores indicate an increased probability of two or more of the adverse pregnancy-related outcomes when the one or more risk scores are at or over 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to one or more reference risk scores. In some embodiments, the one or more risk scores indicate an increased probability of two or more of the adverse pregnancy-related outcomes when the one or more risk scores are at or over 10% compared to one or more one or more reference risk scores. In some embodiments, the one or more risk scores indicate an increased probability of two or more of the adverse pregnancy-related outcomes when the one or more risk scores are at or over 15% compared to one or more reference risk scores. In some embodiments, the one or more risk scores indicate an increased probability of two or more of the adverse pregnancy-related outcomes when the one or more risk scores are at or over 20% compared to one or more reference risk scores. In some embodiments, the one or more risk scores indicate an increased probability of two or more of the adverse pregnancy-related outcomes when the one or more risk scores are at or over 25% compared to one or more reference risk scores. In some embodiments, the one or more reference risk scores are an algorithm-based biomarker scored preterm birth risk.

[0088] In some embodiments, the method for providing preventive intervention of a plurality of adverse pregnancy-related outcomes in a pregnant patient comprises includes (i) measuring in a biological sample obtained from said pregnant patient levels of isolated biomarkers; (ii) calculating one or more risk scores using a reversal value for the isolated biomarkers; and (iii) administering to said pregnant patient, where said one or more risk scores are higher than one or more reference risk scores, a preventive intervention. In some embodiments, the one or more risk scores are connected to the plurality of adverse pregnancy-related outcomes occurring. In some embodiments, a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, indicates an increased probability of at least one adverse pregnancy-related outcome occurring.

[0089] In some embodiments, the method for providing preventive intervention of a plurality of adverse pregnancy -related outcomes in a pregnant patient further includes an initial step of identifying said pregnant human patient as at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium. In some embodiments, the one or more39ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTrisk indicium includes age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections, or any combination thereof. In some embodiments, the one or more risk indicium includes BMI. In some embodiments, the BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2.Adverse Pregnancy-Related Outcomes

[0090] Certain aspects of the methods disclosed herein relate to adverse pregnancy-related outcomes. In some embodiments, the adverse pregnancy-related outcomes include one or more of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) (e.g., predicted GAB being less than 37 weeks), gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal length of stay in hospital (e.g., more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50 or more days), maternal length of stay in hospital (e.g., more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50 or more days), neonatal intensive care unit (NICU) admission, NICU length of stay (e.g., more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50 or more days), neonatal morbidity (e.g., NMI score of 1 or greater, 2 or greater, 3 or greater, or 4), low birthweight, and respiratory distress syndrome (RDS) in the neonate.

[0091] In embodiments, the neonatal morbidity and mortality index score is based on the NICU length of stay and incidence of one or more major neonatal events selected from intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, respiratory distress syndrome, hyaline membrane disease, sepsis, bronchopulmonary dysplasia, and neonatal mortality. In some embodiments, neonatal morbidity index (NMI) score is calculated as follows:40ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT>> >>aAdapted from: Hassan SS, Romero R, Vidyadhari D, el al. PREGNANT Trial. Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial. Ultrasound Obstet Gynecol.2011 Jul;38(l): 18-31. doi: 10.1002 / uog.9017. Epub 2011 Jun 15. PMID: 21472815; PMCID: PMC3482512.Biomarkers and Reversal Values

[0092] Certain aspects of the methods disclosed herein relate to levels of isolated biomarkers and reversal values for calculating one or more risk scores. Such levels of isolated41ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTbiomarkers can be measured using a variety of techniques as disclosed herein, which in some embodiments include measuring levels of isolated biomarkers or surrogate peptides.

[0093] In some embodiments, the isolated biomarkers include one or more biomarkers from Table 1.Table 1: Exemplary isolated biomarkers.

[0094] In some embodiments, the isolated biomarkers include one or more of SHBG, IHNBC, VTNC, ENPP2, PAPP1, PRG2, IBP1, IBP4, NOTUM, ISM2, VGFR1, AOC1, CD 14 or AFAM, or any combination thereof. In some embodiments, the isolated biomarkers include IBP4 and SHBG. In some embodiments, the isolated biomarkers include IBP4 and PAPPl. In some embodiments, the isolated biomarkers include IBP4 and PRG2. In some embodiments, the isolated biomarkers include IBP4 and IBP1. In some embodiments, the isolated biomarkers include IBP4 and NOTUM. In some embodiments, the isolated biomarkers include IBP4 and VGFR1. In some embodiments, the isolated biomarkers include IBP4 and AOC1. In some embodiments, the isolated biomarkers include VTNC and SHBG. In some embodiments, the isolated biomarkers include VTNC and PAPPl. In some embodiments, the isolated biomarkers include VTNC and PRG2. In some embodiments, the isolated biomarkers include VTNC and IBP1. In some embodiments, the isolated biomarkers include VTNC and NOTUM. In some embodiments, the isolated biomarkers include VTNC and VGFR1. In some embodiments, the isolated biomarkers include VTNC and AOC1. In some embodiments, the isolated biomarkers include ENPP2 and SHBG. In some embodiments, the isolated biomarkers include ENPP2 and PAPPl. In some embodiments, the isolated biomarkers include ENPP2 and PRG2. In some42ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTembodiments, the isolated biomarkers include ENPP2 and IBP1. In some embodiments, the isolated biomarkers include ENPP2 and NOTUM. In some embodiments, the isolated biomarkers include ENPP2 and VGFR1. In some embodiments, the isolated biomarkers include ENPP2 and AOC1. In some embodiments, the isolated biomarkers include IHNBC and SHBG. In some embodiments, the isolated biomarkers include IHNBC and PAPPl. In some embodiments, the isolated biomarkers include IHNBC and PRG2. In some embodiments, the isolated biomarkers include IHNBC and IBP1. In some embodiments, the isolated biomarkers include IHNBC and NOTUM. In some embodiments, the isolated biomarkers include IHNBC and VGFR1. In some embodiments, the isolated biomarkers include IHNBC and AOC1.

[0095] In some embodiments, the risk score is calculated using a reversal value for a pair of isolated biomarkers listed in Table 2. In some embodiments, the reversal value is based on a ratio of a numerator biomarker over a denominator biomarker as listed in Table 2.Table 2: Exemplary reversal pairs.43ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0096] In some embodiments, the risk score is calculated using a reversal value for a pair of biomarkers selected from SHBG, IHNBC, VTNC, ENPP2, PAPP1, PRG2, IBP1, IBP4, NOTUM, ISM2, VGFR1, AOC1, CD14 and AFAM, or any combination thereof. In some embodiments, the risk score is calculated using a reversal value for IBP4 and SHBG. In some embodiments, the reversal value is based on a ratio of IBP4 over SHBG. In some embodiments, the risk score is calculated using a reversal value for IBP4 and PAPP1. In some embodiments, the reversal value is based on a ratio of IBP4 over PAPP1. In some embodiments, the risk score is calculated using a reversal value for IBP4 and PRG2. In some embodiments, the reversal value is based on a ratio of IBP4 over PRG2. In some embodiments, the risk score is calculated using a reversal value for IBP4 and IBP1. In some embodiments, the reversal value is based on a ratio of IBP4 over IBP1. In some embodiments, the risk score is calculated using a reversal value for IBP4 and NOTUM. In some embodiments, the reversal value is based on a ratio of IBP4 over NOTUM. In some embodiments, the risk score is calculated using a reversal value for IBP4 and VGFR1. In some embodiments, the reversal value is based on a ratio of IBP4 over VGFR1. In some embodiments, the risk score is calculated using a reversal value for IBP4 and AOC1. In some embodiments, the reversal value is based on a ratio of IBP4 over AOC1. In some embodiments, the risk score is calculated using a reversal value for VTNC and SHBG. In some embodiments, the reversal value is based on a ratio of VTNC over SHBG. In some embodiments, the risk score is calculated using a reversal value for VTNC and PAPP1. In some embodiments, the reversal value is based on a ratio of VTNC over PAPP1. In some embodiments, the risk score is calculated using a reversal value for VTNC and PRG2. In some embodiments, the reversal value is based on a ratio of VTNC over PRG2. In some embodiments, the risk score is calculated using a reversal value for VTNC and IBP1. In some embodiments, the reversal value is based on a ratio of VTNC over IBP1. In some embodiments, the risk score is calculated using a reversal value for VTNC and NOTUM. In some embodiments, the reversal value is based on a ratio of VTNC over NOTUM. In some embodiments, the risk score is calculated using a reversal value for VTNC and VGFR1. In some embodiments, the reversal value is based on a ratio of VTNC over VGFR1. In some44ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTembodiments, the risk score is calculated using a reversal value for VTNC and A0C1. In some embodiments, the reversal value is based on a ratio of VTNC over A0C1. In some embodiments, the risk score is calculated using a reversal value for ENPP2 and SHBG. In some embodiments, the reversal value is based on a ratio of ENPP2 over SHBG. In some embodiments, the risk score is calculated using a reversal value for ENPP2 and PAPP 1. In some embodiments, the reversal value is based on a ratio of ENPP2 over PAPPE In some embodiments, the risk score is calculated using a reversal value for ENPP2 and PRG2. In some embodiments, the reversal value is based on a ratio of ENPP2 over PRG2. In some embodiments, the risk score is calculated using a reversal value for ENPP2 and IBP1. In some embodiments, the reversal value is based on a ratio of ENPP2 over IBP1. In some embodiments, the risk score is calculated using a reversal value for ENPP2 and NOTUM. In some embodiments, the reversal value is based on a ratio of ENPP2 over NOTUM. In some embodiments, the risk score is calculated using a reversal value for ENPP2 and VGFR1. In some embodiments, the reversal value is based on a ratio of ENPP2 over VGFR1. In some embodiments, the risk score is calculated using a reversal value for ENPP2 and AOC 1. In some embodiments, the reversal value is based on a ratio of ENPP2 over AOC1. In some embodiments, the risk score is calculated using a reversal value for IHNBC and SHBG. In some embodiments, the reversal value is based on a ratio of IHNBC over SHBG. In some embodiments, the risk score is calculated using a reversal value for IHNBC and PAPPE In some embodiments, the reversal value is based on a ratio of IHNBC over PAPPE In some embodiments, the risk score is calculated using a reversal value for IHNBC and PRG2. In some embodiments, the reversal value is based on a ratio of IHNBC over PRG2. In some embodiments, the risk score is calculated using a reversal value for IHNBC and IBP1. In some embodiments, the reversal value is based on a ratio of IHNBC over IBP1. In some embodiments, the risk score is calculated using a reversal value for IHNBC and NOTUM. In some embodiments, the reversal value is based on a ratio of IHNBC over NOTUM. In some embodiments, the risk score is calculated using a reversal value for IHNBC and VGFR1. In some embodiments, the reversal value is based on a ratio of IHNBC over VGFR1. In some embodiments, the risk score is calculated using a reversal value for IHNBC and AOC 1. In some embodiments, the reversal value is based on a ratio of IHNBC over AOC 1.

[0097] In some embodiments, the isolated biomarker includes one or more peptide fragments listed in Table 3.45ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTTable 3: Exemplary biomarker peptide fragments.

[0098] In some embodiments, the isolated biomarkers include IBP4, and the biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from SEQ ID NOS: 1-4. In some embodiments, the isolated biomarkers include IBP4, and the biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYI IPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID No: 4).

[0099] In some embodiments, the isolated biomarkers include SHBG, and the biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from SEQ ID NOS: 5-11. In some embodiments, the isolated biomarkers include SHBG, and the biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from IALGGLLFPASNLR (SEQ ID NO: 5), GEDSSTSFCLNGLWAQGQR (SEQ ID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11).

[0100] In addition to the specific biomarkers described herein, 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.Methods of Measuring Biomarkers46ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0101] Certain aspects of the methods disclosed herein relate to methods of measuring levels of isolated biomarkers.

[0102] Any existing, available or conventional separation, detection and quantification methods can be used herein to measure the presence or absence (e.g., readout being present vs. absent; or detectable amount vs. undetectable amount) and / or quantity (e.g., readout being an absolute or relative quantity, such as, for example, absolute or relative concentration) of biomarkers, peptides, polypeptides, proteins and / or fragments thereof and optionally of the one or more other biomarkers or fragments thereof in samples. In some embodiments, detection and / or quantification of one or more biomarkers comprises an assay that utilizes a capture agent. In further embodiments, the capture agent is an antibody, antibody fragment, nucleic acid-based protein binding reagent, small molecule or variant thereof. In additional embodiments, the assay is an enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). In some embodiments, detection and / or quantification of one or more biomarkers further comprises mass spectrometry (MS). In yet further embodiments, the mass spectrometry is co-immunoprecipitation-mass spectrometry (co-IP MS), where coimmunoprecipitation, a technique suitable for the isolation of whole protein complexes is followed by mass spectrometry analysis.

[0103] Generally, any mass spectrometry (MS) technique that can provide precise information on the mass of peptides, and, in some cases, also on fragmentation and / or (partial) amino acid sequence of selected peptides (e.g., in tandem mass spectrometry, MS / MS; or in post source decay, TOF MS), can be used in the methods disclosed herein. Suitable peptide MS and MS / MS techniques and systems are well-known per se (see, e.g., Methods in Molecular Biology, vol. 146: “Mass Spectrometry of Proteins and Peptides”, by Chapman, ed., Humana Press 2000; Biemann 1990. Methods Enzymol 193: 455-79; or Methods in Enzymology, vol.402: “Biological Mass Spectrometry”, by Burlingame, ed., Academic Press 2005) and can be used in practicing the methods disclosed herein. Accordingly, in some embodiments, the disclosed methods comprise performing quantitative MS to measure one or more biomarkers. Such quantitative methods can be performed in an automated (Villanueva, et al., Nature Protocols (2006) l(2):880-891) or semi-automated format. In particular embodiments, MS can be operably linked to a liquid chromatography device (LC-MS / MS or LC-MS) or gas chromatography device (GC-MS or GC -MS / MS). Other methods useful in this context include47ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTisotope-coded affinity tag (ICAT), tandem mass tags (TMT), or stable isotope labeling by amino acids in cell culture (SILAC), followed by chromatography and MS / MS.

[0104] Mass spectrometry assays, instruments and systems suitable for biomarker peptide analysis can include, without limitation, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) MS; MALDI-TOF post-source-decay (PSD); MALDI-TOF / TOF; surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF) MS; electrospray ionization mass spectrometry (ESI-MS); ESI-MS / MS; ESI-MS / (MS)n (n is an integer greater than zero); ESI 3D or linear (2D) ion trap MS; ESI triple quadrupole MS; ESI quadrupole orthogonal TOF (Q-TOF); ESI Fourier transform MS systems; desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCI-MS); APCI-MS / MS; APCI-(MS)n(n is an integer greater than zero); ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS)atmospheric pressure photoionization mass spectrometry (APPI-MS); APPI-MS / MS; and APPI-(MS)n (n is an integer greater than zero). Peptide ion fragmentation in tandem MS (MS / MS) arrangements can be achieved using manners established in the art, such as, e.g., collision induced dissociation (CID). As described herein, detection and quantification of biomarkers by mass spectrometry can involve multiple reaction monitoring (MRM), such as described among others by Kuhn et al. Proteomics 4: 1175-86 (2004). Scheduled multiple-reaction-monitoring (Scheduled MRM) mode acquisition during LC-MS / MS analysis enhances the sensitivity and accuracy of peptide quantitation. Anderson and Hunter, Molecular and Cellular Proteomics 5(4):573 (2006). As described herein, mass spectrometry-based assays can be advantageously combined with upstream peptide or protein separation or fractionation methods, such as for example with the chromatographic and other methods described herein below. As further described herein, shotgun quantitative proteomics can be combined with SRM / MRM-based assays for high-throughput identification and verification of prognostic biomarkers of preterm birth.

[0105] A person skilled in the art will appreciate that a number of methods can be used to determine the amount of a biomarker, including mass spectrometry approaches, such as MS / MS, LC-MS / MS, multiple reaction monitoring (MRM) or SRM and product-ion monitoring (PIM) and also including antibody based methods such as immunoassays such as Western blots, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, and FACS.48ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTAccordingly, in some embodiments, determining the level of the at least one biomarker comprises using an immunoassay and / or mass spectrometry methods. In additional embodiments, the mass spectrometry methods are selected from MS, MS / MS, LC-MS / MS, SRM, PIM, and other such methods that are known in the art. In other embodiments, LC-MS / MS further comprises ID LC-MS / MS, 2D LC-MS / MS or 3D LC-MS / MS. Immunoassay techniques and protocols are generally known to those skilled in the art (Price and Newman, Principles and Practice of Immunoassay, 2nd Edition, Grove’s Dictionaries, 1997; and Gosling, Immunoassays: A Practical Approach, Oxford University Press, 2000.) A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (Self et al., Curr, Opin. BiotechnoL 7:60-65 (1996).

[0106] In further embodiments, the immunoassay is selected from Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay (RIA), dot blotting, and FACS. In certain embodiments, the immunoassay is an ELISA. In yet a further embodiment, the ELISA is direct ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, ELISPOT technologies, and other similar techniques known in the art. Principles of these immunoassay methods are known in the art, for example John R. Crowther, The ELISA Guidebook, 1st ed., Humana Press 2000, ISBN 0896037282. Typically, ELISAs are performed with antibodies but they can be performed with any capture agents that bind specifically to one or more biomarkers of the disclosure and that can be detected. Multiplex ELISA allows simultaneous detection of two or more analytes within a single compartment (e.g., microplate well) usually at a plurality of array addresses (Nielsen and Geierstanger 2004. J Immunol Methods 290: 107-20 (2004) and Ling et al. 2007. Expert Rev Mol Diagn 7: 87-98 (2007)).

[0107] In some embodiments, Radioimmunoassay (RIA) can be used to detect one or more biomarkers in the methods of the disclosure. RIA is a competition-based assay that is well known in the art and involves mixing known quantities of radioactively-labelled (e.g.,125I or13^-labelled) target analyte with antibody specific for the analyte, then adding non-labeled analyte from a sample and measuring the amount of labeled analyte that is displaced (see, e.g., An Introduction to Radioimmunoassay and Related Techniques, by Chard T, ed., Elsevier Science 1995, ISBN 0444821198 for guidance).

[0108] A detectable label can be used in the assays described herein for direct or indirect detection of the biomarkers in the methods of the disclosure. A wide variety of detectable labels49ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTcan be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Those skilled in the art are familiar with selection of a suitable detectable label based on the assay detection of the biomarkers in the methods of the disclosure. Suitable detectable labels include, but are not limited to, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, and the like.

[0109] For mass-spectrometry based analysis, differential tagging with isotopic reagents, e.g., isotope-coded affinity tags (ICAT) or the more recent variation that uses isobaric tagging reagents, iTRAQ (Applied Biosystems, Foster City, Calif.), or tandem mass tags, TMT, (Thermo Scientific, Rockford, IL), followed by multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS / MS) analysis can provide a further methodology in practicing the methods of the disclosure.

[0110] A chemiluminescence assay using a chemiluminescent antibody can be used for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome also can be suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B -phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase, urease, and the like. Detection systems using suitable substrates for horseradish-peroxidase, alkaline phosphatase, and beta-galactosidase are well known in the art.

[0111] A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of125I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer’s instructions. If desired, assays used to practice the disclosure can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.50ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0112] In some embodiments, measuring levels of isolated biomarkers includes subjecting a biological sample to a proteomics workflow. In some embodiments, the proteomics workflow includes mass spectrometry (MS). The proteomics workflow can include any suitable mass spectrometry technique for measuring biomarkers in a sample can be used with the methods provided herein. In some embodiments, the proteomics workflow includes quantification of a stable isotope labeled (SIS) surrogate peptide of the isolated biomarkers. In some embodiments, the MS includes one or more of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS); matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD)); matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI- TOF / TOF); surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS); electrospray ionization mass spectrometry (ESI-MS); electrospray ionization-mass spectrometry / mass spectrometry (ESLMS / MS); electrospray ionization mass spectrometry / mass spectrometry^ (ESI-MS / (MS)n (n is an integer greater than zero), wherein n is an integer greater than zero; electrospray ionization 3D (ESI 3D) or linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS; electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS); electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems); desorption / ionization on silicon (DIOS); secondary ion mass spectrometry (SIMS); atmospheric pressure chemical ionization mass spectrometry (APCI-MS); atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCLMS / MS); atmospheric pressure chemical ionization-mass spectrometry^ (APCI-(MS)n) (n is an integer greater than zero); ion mobility spectrometry (IMS); inductively coupled plasma mass spectrometry (ICP-MS); atmospheric pressure photoionization mass spectrometry (APPL MS); atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPI-MS / MS); and atmospheric pressure photoionization-mass spectrometry^ (APPL(MS)n) (n is an integer greater than zero). In some embodiments, the MS includes co-immunoprecipitationmass spectrometry (co-IPMS). In some embodiments, the MS includes liquid chromatographymass spectrometry (LC-MS). In some embodiments, the MS includes multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

[0113] As described above, chromatography can also be used in practicing the methods of the disclosure. Chromatography encompasses methods for separating chemical substances and generally involves a process in which a mixture of analytes is carried by a moving stream of51ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTliquid or gas (“mobile phase”) and separated into components as a result of differential distribution of the analytes as they flow around or over a stationary liquid or solid phase (“stationary phase”), between the mobile phase and said stationary phase. The stationary phase can be usually a finely divided solid, a sheet of filter material, or a thin film of a liquid on the surface of a solid, or the like. Chromatography is well understood by those skilled in the art as a technique applicable for the separation of chemical compounds of biological origin, such as, e.g., amino acids, proteins, fragments of proteins or peptides, etc.

[0114] Chromatography can be columnar (z.e., wherein the stationary phase is deposited or packed in a column), such as liquid chromatography, high-performance liquid chromatography (HPLC), or ultra-high performance / pressure liquid chromatography (UHPLC). Particulars of chromatography are well known in the art (Bidlingmeyer, Practical HPLC Methodology and Applications, John Wiley & Sons Inc., 1993). Exemplary types of chromatography include, without limitation, high-performance liquid chromatography (HPLC), UHPLC, normal phase HPLC (NP-HPLC), reversed phase HPLC (RP-HPLC), ion exchange chromatography (IEC), such as cation or anion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) including gel filtration chromatography or gel permeation chromatography, chromatofocusing, affinity chromatography such as immuno-affinity, immobilized metal affinity chromatography, and the like. Chromatography, including single-, two- or moredimensional chromatography, can be used as a peptide fractionation method in conjunction with a further peptide analysis method, such as for example, with a downstream mass spectrometry analysis as described elsewhere in this specification.

[0115] Further peptide or polypeptide separation, identification or quantification methods can be used, optionally in conjunction with any of the above-described analysis methods, for measuring biomarkers in the present disclosure. Such methods include, without limitation, chemical extraction partitioning, isoelectric focusing (IEF) including capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary electrochromatography (CEC), and the like, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), free flow electrophoresis (FFE), etc.52ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0116] In some embodiments, measuring levels of isolated biomarkers includes contacting an agent (i.e., a capture agent) that binds to the isolated biomarkers in the biological sample. Exemplary capture agents include, but are not limited to, antibodies, antibody fragments, nucleic acid-based protein binding reagents (e.g., aptamers, Slow Off-rate Modified Aptamers (SOMAmer™)), protein-capture agents, natural ligands (i.e., a hormone for its receptor or vice versa), small molecules or variants thereof. In some embodiments, the capture agent includes an antibody, an antibody fragment, a nucleic acid-based protein binding reagent, a small molecule, or a variant thereof, or any combination thereof. In some embodiments, the contacting is part of an assay selected from the group consisting of enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). In some embodiments, the contacting is followed by MS. In some embodiments, the MS is co-IP MS.

[0117] Capture agents can be configured to specifically bind to a target, in particular a biomarker. Capture agents can include but are not limited to organic molecules, such as polypeptides, polynucleotides and other non-polymeric molecules that are identifiable to a skilled person. In the embodiments disclosed herein, capture agents include any agent that can be used to detect, purify, isolate, or enrich a target, in particular a biomarker. Any art-known affinity capture technologies can be used to selectively isolate and enrich / concentrate biomarkers that are components of complex mixtures of biological media for use in the disclosed methods.

[0118] Antibody capture agents that specifically bind to a biomarker can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986). Antibody capture agents can be any immunoglobulin or derivative thereof, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. Antibody capture agents have a binding domain that is homologous or largely homologous to an immunoglobulin binding domain and can be derived from natural sources, or partly or wholly synthetically produced. Antibody capture agents can be monoclonal or polyclonal antibodies. In some embodiments, an antibody is a single chain antibody. Those of ordinary skill in the art will appreciate that antibodies can be provided in any of a variety of forms including, for example, humanized, partially humanized, chimeric, chimeric humanized, etc. Antibody capture agents can be antibody fragments including, but not limited to, Fab, Fab’,53ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTF(ab’)2, scFv, Fv, dsFv diabody, and Fd fragments. An antibody capture agent can be produced by any means. For example, an antibody capture agent can be enzymatically or chemically produced by fragmentation of an intact antibody and / or it can be recombinantly produced from a gene encoding the partial antibody sequence. An antibody capture agent can comprise a single chain antibody fragment. Alternatively, or additionally, antibody capture agent can comprise multiple chains which are linked together, for example, by disulfide linkages.; and, any functional fragments obtained from such molecules, wherein such fragments retain specificbinding properties of the parent antibody molecule. Because of their smaller size as functional components of the whole molecule, antibody fragments can offer advantages over intact antibodies for use in certain immunochemical techniques and experimental applications.

[0119] Suitable capture agents useful for practicing the disclosure also include aptamers. Aptamers are oligonucleotide sequences that can bind to their targets specifically via unique three-dimensional (3-D) structures. An aptamer can include any suitable number of nucleotides and different aptamers can have either the same or different numbers of nucleotides. Aptamers can be DNA or RNA or chemically modified nucleic acids and can be single stranded, double stranded, or contain double stranded regions, and can include higher ordered structures. An aptamer can also be a photoaptamer, where a photoreactive or chemically reactive functional group is included in the aptamer to allow it to be covalently linked to its corresponding target. Use of an aptamer capture agent can include the use of two or more aptamers that specifically bind the same biomarker. An aptamer can include a tag. An aptamer can be identified using any known method, including the SELEX (systematic evolution of ligands by exponential enrichment), process. Once identified, an aptamer can be prepared or synthesized in accordance with any known method, including chemical synthetic methods and enzymatic synthetic methods and used in a variety of applications for biomarker detection. Liu et al., Curr Med Chem. 18(27):4117-25 (2011). Capture agents useful in practicing the methods of the disclosure also include SOMAmers (Slow Off-Rate Modified Aptamers) known in the art to have improved off-rate characteristics. Brody et al., J Mol Biol. 422(5):595-606 (2012). SOMAmers can be generated using any known method, including the SELEX method.

[0120] It is understood by those skilled in the art that biomarkers can be modified prior to analysis to improve their resolution or to determine their identity. For example, the biomarkers can be subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the biomarkers into a discrete number of fragments are54ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTparticularly useful. The fragments that result from digestion function as a fingerprint for the biomarkers, thereby enabling their detection indirectly. This is particularly useful where there are biomarkers with similar molecular masses that might be confused for the biomarker in question. Also, proteolytic fragmentation is useful for high molecular weight biomarkers because smaller biomarkers are more easily resolved by mass spectrometry. In another example, biomarkers can be modified to improve detection resolution. For instance, neuraminidase can be used to remove terminal sialic acid residues from glycoproteins to improve binding to an anionic adsorbent and to improve detection resolution. In another example, the biomarkers can be modified by the attachment of a tag of particular molecular weight that specifically binds to molecular biomarkers, further distinguishing them. Optionally, after detecting such modified biomarkers, the identity of the biomarkers can be further determined by matching the physical and chemical characteristics of the modified biomarkers in a protein database (e.g., SwissProt).

[0121] It is further appreciated in the art that biomarkers in a sample can be captured on a substrate for detection. Traditional substrates include antibody-coated 96-well plates or nitrocellulose membranes that are subsequently probed for the presence of the proteins. Alternatively, protein-binding molecules attached to microspheres, microparticles, microbeads, beads, or other particles can be used for capture and detection of biomarkers. The proteinbinding molecules can be antibodies, peptides, peptoids, aptamers, small molecule ligands or other protein-binding capture agents attached to the surface of particles. Each protein-binding molecule can include unique detectable label that is coded such that it can be distinguished from other detectable labels attached to other protein-binding molecules to allow detection of biomarkers in multiplex assays. Examples include, but are not limited to, color-coded microspheres with known fluorescent light intensities (see, e.g., microspheres with xMAP technology produced by Luminex (Austin, Tex.); microspheres containing quantum dot nanocrystals, for example, having different ratios and combinations of quantum dot colors (e.g., Qdot nanocrystals produced by Life Technologies (Carlsbad, Calif.); glass coated metal nanoparticles (see, e.g, SERS nanotags produced by Nanoplex Technologies, Inc. (Mountain View, Calif.); barcode materials (see, e.g., sub-micron sized striped metallic rods such as Nanobarcodes produced by Nanoplex Technologies, Inc.), encoded microparticles with colored bar codes (see, e.g., CellCard produced by Vitra Bioscience, vitrabio.com), glass microparticles with digital holographic code images (see, e.g., CyVera microbeads produced by Illumina (San55ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTDiego, Calif.); chemiluminescent dyes, combinations of dye compounds; and beads of detectably different sizes.

[0122] In another aspect, biochips can be used for capture and detection of the biomarkers of the disclosure. Many protein biochips are known in the art. These include, for example, protein biochips produced by Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward, Calif.) and Phylos (Lexington, Mass.). In general, protein biochips comprise a substrate having a surface. A capture reagent or adsorbent is attached to the surface of the substrate. Frequently, the surface comprises a plurality of addressable locations, each of which location has the capture agent bound there. The capture agent can be a biological molecule, such as a polypeptide or a nucleic acid, which captures other biomarkers in a specific manner. Alternatively, the capture agent can be a chromatographic material, such as an anion exchange material or a hydrophilic material. Examples of protein biochips are well known in the art.

[0123] In some embodiments, measuring levels of isolated biomarkers includes measuring levels of one or more biomarkers from Table 1. In some embodiments, measuring levels of isolated biomarkers includes measuring levels of one or more peptide fragments of one or more biomarkers from Table 1. In some embodiments, measuring levels of isolated biomarkers includes measuring levels of one or more peptide fragments listed in Table 3.

[0124] The quantitation of biomarkers in a biological sample can be determined, without limitation, by the methods described above as well as any other method known in the art. The quantitative data thus obtained is then subjected to an analytic classification process. In such a process, the raw data is manipulated according to an algorithm, where the algorithm has been pre-defined by a training set of data, for example as described in the examples provided herein. An algorithm can utilize the training set of data provided herein or can utilize the guidelines provided herein to generate an algorithm with a different set of data.Risk Scores

[0125] Certain aspects of the methods disclosed herein relate to risk scores for determining the probability of an adverse pregnancy-related outcome. In some embodiments, the risk score is calculated using the reversal value for the panel of biomarkers. In some embodiments, the risk scores are used in a predictive model for adverse pregnancy-related outcomes. In some embodiments, the predictive model includes the comparison of a risk score to a reference risk score.56ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0126] Some methods of the disclosure involve (1) detecting the expression levels of a panel of isolated biomarkers (e.g., IBP4 and SHBG) in a biological sample from the individual; (2) providing a test biomarker score corresponding to the expression levels of said panel; (3) providing at least one test clinical score representing at least one clinical variable; (4) combining the test biomarker score and the at least one test clinical score to yield a test combined score; and (5) diagnosing the individual as having either (a) an increased likelihood of an adverse pregnancy-related outcome based at least in part on said test combined score equaling or exceeding a first reference score or (b) no increased likelihood of preterm birth based at least in part on said test combined score not equaling or exceeding a second reference score.

[0127] In some embodiments, the one or more test scores indicate an increased likelihood of an adverse pregnancy-related outcome or likelihood of responsiveness to a prophylactic treatment regimen when the one or more test scores are at or over one or more reference risk scores. In some embodiments, the one or more test scores indicates an increased likelihood of an adverse pregnancy-related outcome or likelihood of responsiveness to a prophylactic treatment regimen when the one or more test scores are at or over 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to one or more reference scores. In some embodiments, the one or more test scores indicate an increased likelihood of an adverse pregnancy-related outcome or likelihood of responsiveness to a prophylactic treatment regimen when the one or more test scores are a multiple of the one or more one or more reference scores (e.g., 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 or more times the reference score). In some embodiments, the one or more reference scores are an algorithm-based biomarker scored risk of an adverse pregnancy-related outcome.

[0128] As those skilled in the art can appreciate, such comparison can be a direct comparison to the reference score or an indirect comparison where the reference score has been incorporated into the predictive model. In further embodiments, analyzing a measurable feature to determine the probability for adverse pregnancy-related outcomes in a pregnant human patient encompasses one or more of a linear discriminant analysis model, a support vector machine classification algorithm, a recursive feature elimination model, a prediction analysis of microarray model, a logistic regression model, a CART algorithm, a flex tree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, a machine learning algorithm,57ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTa penalized regression method, or a combination thereof. In particular embodiments, the analysis comprises logistic regression.

[0129] An analytic classification process can use any one of a variety of statistical analytic methods to manipulate the quantitative data and provide for classification of the sample. Examples of useful methods include linear discriminant analysis, recursive feature elimination, a prediction analysis of microarray, a logistic regression, a CART algorithm, a FlexTree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, machine learning algorithms; etc.

[0130] In some embodiments, a generalized linear model (GLM) adds multiple variables, each assigned a coefficient, to yield a score. Such models will typically be of the general formula SCORE = AX + BY + CZ + [... ], where A, B, C, etc. are coefficients and X, Y, Z, etc. are variables. In some embodiments, when a variable is a protein or peptide, the variable value is the concentration of the protein / peptide in a biological sample. Such a model can be fit to outcomes of preterm birth or preeclampsia or gestational diabetes.

[0131] Importantly, this approach can be broadened to expand or reduce the list of clinical / demographic factors and proteins / peptides. It can also be fitted, using the same methodology, to different outcome groups such as restriction to just preterm birth or expanded to also include gestational hypertension. Well-established statistical techniques can be used to optimize the GLM to avoid overfitting. Table 4 shows an example of a model that combines seven clinical / demographic factors with proteomic variables of up to 40 proteins / peptides. The specific clinical / demographic factors and peptides are listed in Table 4, together with the coefficients assigned to each such variable in the linear model. This model can be used to predict adverse pregnancy outcomes as disclosed herein, the performance of which is reported in Example 1 of the present disclosure. In some embodiments, any panel of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, or 40) proteins / peptides from Table 4 is combined with any one or more clinical variables (1, 2, 3, 4, 5, 6, or 7) in Table 4 to create a predictor of adverse pregnancy outcomes as disclosed herein.Table 4: Representative Model Combining Seven Clinical / Demographic Factors with Proteomic Variables of Up To 40 Proteins / Peptides58ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT59ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0132] Classification can be made according to predictive modeling methods that set a threshold for determining the probability that a sample belongs to a given class. The probability may be at least 50%, or at least 60%, or at least 70%, or at least 80% or higher. Classifications also can be made by determining whether a comparison between an obtained dataset and a reference dataset yields a statistically significant difference. If so, then the sample from which the dataset was obtained is classified as not belonging to the reference dataset class. Conversely, if such a comparison is not statistically significantly different from the reference dataset, then the sample from which the dataset was obtained is classified as belonging to the reference dataset class.

[0133] The predictive ability of a model can be evaluated according to its ability to provide a quality metric, e.g., AUROC (area under the ROC curve) or accuracy, of a particular value, or range of values. Area under the curve measures are useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest. In some embodiments, a desired quality threshold is a predictive model that will classify a sample with an accuracy of at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or higher. As an alternative measure, a desired quality threshold can refer to a predictive model that will classify a sample with an AUC of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[0134] As is known in the art, the relative sensitivity and specificity of a predictive model can be adjusted to favor either the selectivity metric or the sensitivity metric, where the two metrics have an inverse relationship. The limits in a model as described above can be adjusted to provide a selected sensitivity or specificity level, depending on the particular requirements60ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTof the test being performed. One or both of sensitivity and specificity can be at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[0135] The raw data can be initially analyzed by measuring the values for each biomarker, usually in triplicate or in multiple triplicates. The data can be manipulated, for example, raw data can be transformed using standard curves, and the average of triplicate measurements used to calculate the average and standard deviation for each patient. These values can be transformed before being used in the models, e.g., log-transformed, Box-Cox transformed (Box and Cox, Royal Stat. Soc„ Series B, 26:211-246(1964). The data are then input into a predictive model, which will classify the sample according to the state. The resulting information can be communicated to a patient or health care provider.

[0136] To generate a predictive model for an adverse pregnancy -related outcome, a robust data set, comprising known control samples and samples corresponding to the preterm birth classification of interest is used in a training set. A sample size can be selected using generally accepted criteria. As discussed above, different statistical methods can be used to obtain a highly accurate predictive model. Examples of such analysis are provided in Example 2.

[0137] In one embodiment, hierarchical clustering is performed in the derivation of a predictive model, where the Pearson correlation is employed as the clustering metric. One approach is to consider a preterm birth dataset as a “learning sample” in a problem of “supervised learning.” CART is a standard in applications to medicine (Singer, Recursive Partitioning in the Health Sciences, Springer (1999)) and can be modified by transforming any qualitative features to quantitative features; sorting them by attained significance levels, evaluated by sample reuse methods for Hotelling’s T2statistic; and suitable application of the lasso method. Problems in prediction are turned into problems in regression without losing sight of prediction, indeed by making suitable use of the Gini criterion for classification in evaluating the quality of regressions.

[0138] This approach led to what is termed FlexTree (Huang, Proc. Nat. Acad. Sci. U.S.A 101:10529-10534(2004)). FlexTree performs very well in simulations and when applied to multiple forms of data and is useful for practicing the claimed methods. Software automating FlexTree has been developed. Alternatively, LARTree or LART can be used (Turnbull (2005) Classification Trees with Subset Analysis Selection by the Lasso, Stanford University). The name reflects binary trees, as in CART and FlexTree; the lasso, as has been noted; and the61ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTimplementation of the lasso through what is termed LARS by Efron et al. (2004) Annals of Statistics 32:407-451 (2004). See, also, Huang et al.., Proc. Natl. Acad. Sci. USA.101(29): 10529-34 (2004). Other methods of analysis that can be used include logic regression. One method of logic regression Ruczinski, Journal of Computational and Graphical Statistics 12:475-512 (2003). Logic regression resembles CART in that its classifier can be displayed as a binary tree. It is different in that each node has Boolean statements about features that are more general than the simple “and” statements produced by CART.

[0139] Another approach is that of nearest shrunken centroids (Tibshirani, Proc. Natl. Acad. Sci. U.S.A 99:6567-72(2002)). The technology is k-means-like, but has the advantage that by shrinking cluster centers, one automatically selects features, as is the case in the lasso, to focus attention on small numbers of those that are informative. The approach is available as PAM software and is widely used. Two further sets of algorithms that can be used are random forests (Breiman, Machine Learning 45:5-32 (2001)) and MART (Hastie, The Elements of Statistical Learning, Springer (2001)). These two methods are known in the art as “committee methods,” that involve predictors that “vote” on outcome.

[0140] To provide significance ordering, the false discovery rate (FDR) can be determined. First, a set of null distributions of dissimilarity values is generated. In one embodiment, the values of observed profiles are permuted to create a sequence of distributions of correlation coefficients obtained out of chance, thereby creating an appropriate set of null distributions of correlation coefficients (Tusher et al., Proc. Natl. Acad. Sci. U.S.A 98, 5116-21 (2001)). The set of null distribution is obtained by: permuting the values of each profile for all available profiles; calculating the pair-wise correlation coefficients for all profile; calculating the probability density function of the correlation coefficients for this permutation; and repeating the procedure for N times, where N is a large number, usually 300. Using the N distributions, one calculates an appropriate measure (mean, median, etc.) of the count of correlation coefficient values that their values exceed the value (of similarity) that is obtained from the distribution of experimentally observed similarity values at given significance level.

[0141] The FDR is the ratio of the number of the expected falsely significant correlations (estimated from the correlations greater than this selected Pearson correlation in the set of randomized data) to the number of correlations greater than this selected Pearson correlation in the empirical data (significant correlations). This cut-off correlation value can be applied to the correlations between experimental profiles. Using the aforementioned distribution, a level62ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTof confidence is chosen for significance. This is used to determine the lowest value of the correlation coefficient that exceeds the result that would have obtained by chance. Using this method, one obtains thresholds for positive correlation, negative correlation or both. Using this threshold(s), the user can filter the observed values of the pair wise correlation coefficients and eliminate those that do not exceed the threshold(s). Furthermore, an estimate of the false positive rate can be obtained for a given threshold. For each of the individual “random correlation” distributions, one can find how many observations fall outside the threshold range. This procedure provides a sequence of counts. The mean and the standard deviation of the sequence provide the average number of potential false positives and its standard deviation.

[0142] In an alternative analytical approach, variables chosen in the cross-sectional analysis are separately employed as predictors in a time-to-event analysis (survival analysis), where the event is the occurrence of preterm birth, and subjects with no event are considered censored at the time of giving birth. Given the specific adverse pregnancy-related outcome, the random lengths of time each patient will be observed, and selection of proteomic and other features, a parametric approach to analyzing survival can be better than the widely applied semi-parametric Cox model. A Weibull parametric fit of survival permits the hazard rate to be monotonically increasing, decreasing, or constant, and also has a proportional hazards representation (as does the Cox model) and an accelerated failure-time representation. All the standard tools available in obtaining approximate maximum likelihood estimators of regression coefficients and corresponding functions are available with this model.

[0143] In addition, the Cox models can be used, especially since reductions of numbers of covariates to manageable size with the lasso will significantly simplify the analysis, allowing the possibility of a nonparametric or semi-parametric approach to prediction of time to preterm birth. These statistical tools are known in the art and applicable to all manner of proteomic data. A set of biomarker, clinical and genetic data that can be easily determined, and that is highly informative regarding the probability for preterm birth and predicted time to a preterm birth event in said pregnant human patient is provided. Also, algorithms provide information regarding the probability for preterm birth in the pregnant human patient.

[0144] As will be understood by those skilled in the art, an analytic classification process can use any one of a variety of statistical analytic methods to manipulate the quantitative data and provide for classification of the sample. Examples of useful methods include, without limitation, linear discriminant analysis, recursive feature elimination, a prediction analysis of63ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTmicroarray, a logistic regression, a CART algorithm, a FlexTree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, and machine learning algorithms. Various methods are used in a training model. The selection of a subset of markers can be for a forward selection or a backward selection of a marker subset. The number of markers can be selected that will optimize the performance of a model without the use of all the markers. One way to define the optimum number of terms is to choose the number of terms that produce a model with desired predictive ability (e.g., an AUC>0.75, or equivalent measures of sensitivity / specificity) that lies no more than one standard error from the maximum value obtained for this metric using any combination and number of terms used for the given algorithm.Preventive Interventions

[0145] Certain aspects of the methods disclosed herein relate to interventions aimed at preventing one or more adverse pregnancy -related outcomes.

[0146] In some embodiments, the preventive intervention is for a pregnant human patient identified as having an increased probability of preterm birth (PTB). In such a case, in some embodiments, the preventive intervention for the pregnant human patient includes one or more of administration of aspirin, a progesterone treatment, and transvaginal cervical length screenings. In some embodiments, the progesterone treatment includes one or more of progestogen, 17-a hydroxyprogesterone caproate, and vaginal progesterone. In some embodiments, 17-a hydroxyprogesterone caproate is administered as an injection. In some embodiments, vaginal progesterone is administered in gel form. In some embodiments, a preventive intervention for the pregnant human patient includes administration of tocolytics to delay labor. In some embodiments, a preventive intervention for the pregnant human patient includes administration of low-dose aspirin. In some embodiments, a preventive intervention for the pregnant human patient includes, separately or in addition to a medical intervention as described above, weekly nurse telephonic support, bed rest and / or reduced physical activity.

[0147] In some embodiments, the preventive intervention is for a pregnant human patient identified as having an increased probability of preeclampsia (PE). In such a case, in some embodiments, the preventive intervention for the pregnant human patient includes one or more of administration of magnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity and / or bed rest.64ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0148] In some embodiments, the preventive intervention is for a pregnant human patient identified as having an increased probability of predicted gestational age at birth GAB being less than 37 weeks. In such a case, in some embodiments, the preventive intervention for the pregnant human patient includes administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity. In some embodiments, the preventive intervention is for the neonate for a pregnant human patient identified as having an increase probability of predicted gestational age at birth GAB being less than 37 weeks. In such a case, in some embodiments, the preventive intervention for the neonate includes use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and / or a stay in a neonatal intensive care unit (NICU).

[0149] In some embodiments, the preventive intervention is for a pregnant human patient identified as having an increased probability of gestational diabetes mellitus (GDM). In such a case, in some embodiments, the preventive intervention for the pregnant human patient includes one or more of administration of insulin, an oral hypoglycemic agent (e.g., metformin), medical nutrition therapy, increased physical activity, and / or weight management.

[0150] In some embodiments, the preventive intervention is for a pregnant human patient identified as having an increased probability of gestational hypertension (gHTN). In such a case, in some embodiments, the preventive intervention for the pregnant human patient includes one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication (e.g., methyldopa, labetalol, nifedipine, hydralazine, prazosin, or clonidine), fetal monitoring, hospitalization, and / or bedrest.

[0151] In some embodiments, the preventive intervention is for a neonate carried by a pregnant human patient or the pregnant human patient herself identified as having an increased probability of low birthweight. In such a case, in some embodiments, the preventive intervention for the pregnant human patient includes one or more of administration of nutritional supplements. In some embodiments, the therapeutic treatment for the neonate includes use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and / or a stay in a NICU.

[0152] In some embodiments, the preventive intervention is for a neonate carried by a pregnant human patient identified as having an increased probability of delivering a neonate with respiratory distress syndrome (RDS). In such a case, some embodiments, the preventive65ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTintervention for the neonate includes one or more of surfactant therapy, oxygen therapy, continuous positive airway pressure, mechanical ventilation, nutritional support, and / or prone positioning.Resource Allocation

[0153] Certain aspects of the methods disclosed herein relate to allocation of hospital resources.

[0154] In some embodiments, the methods described herein further includes allocating hospital resources to a pregnant patient that has been identified to have an increased probability of at least one adverse pregnancy-related outcome occurring. The hospital resource allocation can be part of a hospital resource allocation scheme that is informed by the determination that a pregnant patient has an increase probability of at least one, at least two, at least three, at least four, or more adverse pregnancy-related outcome of the plurality of pregnancy-related outcomes described herein. The hospital resource allocation scheme can also include a hospital or physician tailoring clinical strategies for the pregnant human patient, or the neonate carried by the pregnant human patient, to include one or more preventive interventions described herein. For instance, the hospital resource allocation scheme may include allocation of a hospital bed to a pregnant patient that has been identified as having increased likelihood of preeclampsia. Similarly, the hospital resource allocation scheme can include increased medical team and a NICU stay for a neonate carried by a pregnant patient identified as having increased likelihood of low birthweight birth. In some embodiments, the hospital resource allocation scheme can include increased monitoring of a pregnant patient, or a neonate carried by the pregnant patient, where the pregnant patient has been determined to have an increased probability of a plurality of adverse pregnancy-related outcomes (e.g., as determined by the methods described herein). The hospital resource allocation scheme can be further modified in response to characteristic of the patient and / or one or more clinical factors (e.g., clinical factors described herein). The changes in hospital resource allocation can be ward-specific or a hospital-wide change in allocation. The ability to determine the likelihood of different adverse pregnancy-related outcomes provides for customization of preventive interventions commonly used for the determined increased probability of at least one, at least two, at least three, at least four, or more adverse pregnancy-related outcome of the plurality of pregnancy-related outcomes described herein, thereby maximizing the intervention that is used and resulting in a cost-effective allocation of resources.66ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT

[0155] In some embodiments, provided herein is a method of allocating hospital resources including: receiving a determination of an increased probability of at least one adverse pregnancy-related outcome for a pregnant patient, or a neonate carried by the pregnant patient, by, e.g., a method for determining the probability of a plurality of adverse pregnancy -related outcomes in a pregnant patient described herein; and modifying a hospital resource allocation scheme based, at least in part, on the increased probability of the at least one adverse-pregnancy related outcome. In some embodiments, modifying a hospital resource allocation scheme includes allocating hospital resources to accommodate the increased probability of the at least one adverse pregnancy-related outcome in the pregnant patient, or the neonate carried by the pregnant patient. In some embodiments, the hospital resources include hospital beds or rooms, hospital equipment, or increased monitoring by a physician. In some embodiments, modifying a hospital resource allocation scheme includes implementation of one or more preventive interventions described herein.

[0156] In some embodiments, the method of allocating hospital resources further includes communicating the increased probability of the at least one adverse pregnancy -related outcome to a hospital or physician. In some embodiments, the increased probability of the at least one adverse pregnancy-related outcome can be communicated to the hospital or physician as part of a report. The report can be a printed report or digital. In some embodiments, the report further includes demographic information for the pregnant patient and / or information about one or more clinical factors (e.g., clinical factors described herein) for the pregnant patient. In some embodiments, the report further includes a recommendation for modifying the hospital resource allocation scheme. For examples, the report can include recommendations on one or more preventive interventions for the at least one adverse pregnancy-related outcome, assignment of a hospital bed to the patient, increased monitoring by a physician, or additional diagnostic tests. In some embodiments, after communicating the increased probability of the at least one adverse pregnancy-related outcome to the hospital or physician, the hospital or physician modifies a hospital resource allocation scheme based, at least in part, on the increased probability of the at least one pregnancy -related outcome in the pregnant patient.Biological Samples

[0157] Certain aspects of the methods disclosed herein relate to biological samples. The biological sample can include any biological sample type suitable for use with the methods disclosed herein. In some embodiments, the biological sample is whole blood, plasma, serum,67ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTamniotic fluid, vaginal secretions, saliva, or urine. In some embodiments, the biological sample is whole blood, plasma or serum.

[0158] The biological sample may be obtained from the pregnant patient at any suitable estimated gestational age. In some embodiments, the pregnant human patient is between 17 and 28 weeks of gestation at the time the biological sample is collected. In other embodiments, the pregnant human patient is between 16 and 29 weeks, between 17 and 28 weeks, between 18 and 27 weeks, between 19 and 26 weeks, between 20 and 25 weeks, between 21 and 24 weeks, or between 22 and 23 weeks of gestation at the time the biological sample is collected. In further embodiments, the pregnant human patient is between about 17 and 22 weeks, between about 16 and 22 weeks between about 22 and 25 weeks, between about 13 and 25 weeks, between about 26 and 28, or between about 26 and 29 weeks of gestation at the time the biological sample is collected. Accordingly, the estimated gestational age of a pregnant human patient at the time the biological sample is collected can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 weeks. In particular embodiments, the biological sample is collected between 19 and 21 weeks of estimated gestational age. In particular embodiments, the biological sample is collected between 19 and 22 weeks of estimated gestational age. In particular embodiments, the biological sample is collected between 19 and 21 weeks of estimated gestational age. In particular embodiments, the biological sample is collected between 19 and 22 weeks of estimated gestational age. In particular embodiments, the biological sample is collected at 18 weeks of estimated gestational age.Patients

[0159] Certain aspects of the methods disclosed herein relate to patients, and in particular pregnant females and neonates. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0160] In some embodiments, the subject is a pregnant female. In some embodiments, the patient is a pregnant human female. In some embodiments, the pregnant human female is between 17 and 28 weeks of gestation. In other embodiments, the pregnant human female is between 16 and 29 weeks, between 17 and 28 weeks, between 18 and 27 weeks, between 19 and 26 weeks, between 20 and 25 weeks, between 21 and 24 weeks, or between 22 and 23 weeks of gestation. In further embodiments, the pregnant human female is between about 17 and 22 weeks, between about 16 and 22 weeks between about 22 and 25 weeks, between about68ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT13 and 25 weeks, between about 26 and 28, or between about 26 and 29 weeks of gestation at the time the biological sample is collected.

[0161] In some embodiments, the pregnant human female has not previously been pregnant.

[0162] In some embodiments, the pregnant human female has previously been pregnant. In some embodiments, the pregnant human female has a history of previous low birth weight or preterm delivery. In some embodiments, the pregnant human female has a history of multiple 2nd trimester spontaneous abortions. In some embodiments, the pregnant human female has a history of prior first trimester induced abortion.

[0163] In some embodiments, the pregnant human female has a history of infertility.

[0164] In some embodiments, the pregnant human female has a history of one or more of nulliparity, placental abnormalities, cervical and / or uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero di ethyl stilbestrol exposure, gravidity, diabetes, hypertension, hypothyroidism, asthma, tobacco use, or urogenital infections.

[0165] In some embodiments, the pregnant human female has short statute.

[0166] In some embodiments, the pregnant human female has a low pre-pregnancy weight or low BMI. In some embodiments, the pregnant human female has a BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2.Kits

[0167] Certain aspects relate to kits for used in the methods disclosed herein. The kit can include one or more agents for detection of biomarkers, a container for holding a biological sample isolated from a pregnant human patient; and printed instructions for reacting agents with the biological sample or a portion of the biological sample to detect the presence or amount of the isolated biomarkers in the biological sample. The agents can be packaged in separate containers. The kit can further comprise one or more control reference samples and reagents for performing an immunoassay.

[0168] The kit can comprise one or more containers for compositions contained in the kit. Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be 69ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTformed from a variety of materials, including glass or plastic. The kit can also comprise a package insert containing written instructions for methods of determining probability of adverse pregnancy -related outcomes in a pregnant human patient.EXAMPLES

[0169] In order that this disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the disclosure in any manner.Example 1: Development of a Predictor for Complications of Pregnancy

[0170] Some of the main complications of pregnancy include preterm birth, preeclampsia, gestational diabetes, and gestational hypertension, which occur collectively in about 25% of all pregnancies. Early detection of these complications before symptoms manifest allows for their effective management. Current methods for the early detection of these complications typically consist of clinical risk factors such as high body mass index (BMI) or a complication in a prior pregnancy. However, these methods are ineffective for first time mothers (40% of pregnancies) and for most other pregnancies since the majority of complications have no such risk factors. Additionally, risk factors such as high BMI can be ineffective in populations such as the United States where high BMI is prevalent in the population.

[0171] The research reported in this Example 1 shows that a predictor incorporating proteomic biomarker measurements was able to detect increased risk of complications early in pregnancy to allow for better stratification of pregnancies than would be possible using clinical risk factors alone. The stratification of pregnancies achieved using the predictor incorporating the proteomic markers reduced both false positives and false negatives, which may allow for improved clinical outcomes, more efficient use of resources, and reduced costs.

[0172] This Example 1 describes the development of a predictor that combines clinical and demographic risk factors with a proteomic biomarker assay to more accurately identify which pregnancies are at risk and which are not at risk for complications compared to existing methods.

[0173] Serum samples from 976 patients were tested using the proteomic biomarker assay to detect levels of IBP4 and SHBG as described in Saade et al. (“Development and validation of a spontaneous preterm delivery predictor in asymptomatic women.” Am J Obstet Gynecol,70ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT2016. 214(5): p. 633 el-633 e24). Clinical and demographic factors were known for each patient and were combined with reversal scores derived from such assay, to yield a combined score trained to predict the risk of adverse pregnancy outcomes.

[0174] Clinical and demographic factors incorporated into the model included: maternal age, BMI, race, gravida, prior spontaneous preterm birth (sPTB), prior preeclampsia, smoking, drug use, prior miscarriage, and prior loss fetus after 20 weeks.

[0175] A generalized linear model was used to integrate clinical and demographic factors with proteomic reversal scores.

[0176] Adverse pregnancy outcomes predicted (i.e., increased risk of any one of these outcomes) included preeclampsia, preterm birth, gestational diabetes, C-section, maternal length of stay in hospital (MLOSH), neonatal length of stay in the hospital, and neonatal length of stay in NICU.

[0177] Thresholds for calling a patient “high risk” were assessed in terms of performance (sensitivity (sens), specificity, positive predictive value (PPV), negative predictive value (NPV), and screen positive rate (SPR)).

[0178] The performance of the combined predictor (clinical factors plus proteomic assay results, solid lines denoted “assay” in FIG. 1), as compared to clinical factors alone (corresponding dotted lines in FIG. 1), is shown in FIG. 1. The predictor identified both pregnancies at high risk (PPV > 70%, SPR < 10%) and at low risk (NPV > 98%, SNR > 40%). Pregnancies identified as neither high nor low risk were not distinguishable from usual risk (i.e., standard care paradigms can be applied). Combining proteomic reversal score with clinical factors in the predictor allowed for the identification of specific complication(s) to be addressed for high-risk patients, such as patients with gestational diabetes or having had a previous preterm birth. For all predictor thresholds, but especially from 0.5 to 0.7, the combined predictor was more sensitive and accurate (PPV), at approximately the same screen positive rate, than clinical factors alone.

[0179] As shown in FIG. 2, the performance profile of the combined predictor of the present disclosure (solid lines, denoted “assay” in FIG. 2) was substantially better than the current practice of clinical and demographic factors alone (corresponding dotted lines, denoted “risk factors” in FIG. 2) for the identification of pregnancies with (and without) multiple71ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTadverse outcomes (i.e., for identifying pregnancies at increased risk of such outcomes). As one illustrative example, the predictor identified more than 5 times as many pregnancies with long hospital stays for the mother and baby as well as unplanned C-sections. In FIG. 2, the x-axis is the predictor score ranging from 0 to 1 and the y-axis is number of occurrences of adverse pregnancy outcomes. FIG. 2 illustrates that, when clinical and demographic factors were combined with measurements from the proteomic biomarker assay (i.e., a combined predictor), the result was a higher detection rate than that achieved using clinical and demographic factors alone. At a combined predictor score 0.50 to 0.75 there was a distinct separation between the detection rate for the combined predictor over clinical and demographic factors alone for maternal and neonatal hospital stay as well as for preterm C-sections.EQUIVALENTS AND SCOPE, INCORPORATION BY REFERENCE

[0180] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is understood that modifications which do not substantially affect the activity of the various embodiments of this disclosure are also provided within the description of the disclosure provided herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0181] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or all of the group members, are present in, employed in, or otherwise relevant to a given product or process.

[0182] Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is 72ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTto be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0183] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the embodiments, is / are referred to as that includes particular elements, features, steps, etc., certain embodiments of the disclosure or aspects of the embodiments consist, or consist essentially of, such elements, features, steps, etc. Thus, for each embodiment of the disclosure that includes one or more elements, features, steps, etc., the disclosure also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[0184] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0185] In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0186] Throughout this disclosure various publications, patents, and sequence database entries are mentioned. The disclosures of these publications, patents, and sequence database73ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTentries, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0187] Although the disclosure has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the scope of the disclosure. Accordingly, the above examples are intended to illustrate but not limit the present disclosure.74ACTIVE 718780158v1

Claims

Attorney Docket No. 203123-030002 / PCTCLAIMSWhat is claimed is:

1. A method of determining the probability of a plurality of adverse pregnancy-related outcomes in a pregnant human patient, the method comprising(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 and SHBG,wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to the plurality of adverse pregnancy-related outcomes occurring,wherein a higher risk score in the pregnant human patient compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, indicates an increased probability of at least one adverse pregnancy -related outcome occurring, andwherein the plurality of adverse pregnancy-related outcomes comprise two or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

2. The method of claim 1, wherein the method further comprises combining said reversal value with one or more clinical factors to calculate said one or more risk scores for the plurality of adverse pregnancy-related outcomes occurring, wherein said one or more clinical factors is selected from the group consisting of age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic75ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTdiabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy.

3. The method of claim 1 or 2, further comprising an initial step of identifying said pregnant human patient as potentially at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium.

4. The method of claim 3, wherein said one or more risk indicium is selected from the group consisting of age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortions, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and / or uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections.

5. The method of claim 4, wherein said one or more risk indicium comprises BMI.

6. The method of claim 5, wherein said BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2.

7. The method of any one of claims 1-6, wherein said biological sample is selected from the group consisting of whole blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine.

8. The method of claim 7, wherein said biological sample is whole blood, plasma, or serum.

9. The method of any one of claims 1-8, wherein said biological sample is obtained between 19 and 21 weeks of estimated gestational age.

10. The method of any one of claims 1-9, wherein said measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

11. The method of claim 10, wherein said proteomics workflow comprises quantification of a stable isotope labeled (SIS) surrogate peptide of said isolated biomarkers.76ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT12. The method of claim 10, wherein said MS is selected from the group consisting of:a) matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS);b) matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD));c) matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDL TOF / TOF);d) surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS);e) electrospray ionization mass spectrometry (ESI-MS);f) electrospray ionization-mass spectrometry / mass spectrometry (ESI-MS / MS); g) electrospray ionization mass spectrometry / mass spectrometry^ (ESI-MS / (MS)n, wherein n is an integer greater than zero;h) electrospray ionization 3D (ESI 3D) or linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS;i) electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS);j) electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); k) electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems);l) desorption / ionization on silicon (DIOS);m) secondary ion mass spectrometry (SIMS);n) atmospheric pressure chemical ionization mass spectrometry (APCLMS); o) atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCLMS / MS);p) atmospheric pressure chemical ionization-mass spectrometry^ (APCI-(MS)n), wherein n is an integer greater than zero;q) ion mobility spectrometry (IMS);r) inductively coupled plasma mass spectrometry (ICP-MS);s) atmospheric pressure photoionization mass spectrometry (APPLMS); t) atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPI-MS / MS); and77ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTu) atmospheric pressure photoionization-mass spectrometry^ (APPI-(MS)n), wherein n is an integer greater than zero.

13. The method of claim 10, wherein said MS comprises co-immunoprecipitationmass spectrometry (co-IP MS).

14. The method of claim 10, wherein said MS comprises liquid chromatographymass spectrometry (LC-MS).

15. The method of claim 10, wherein said MS comprises multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

16. The method of any one of claims 1-15, further comprising communicating said adverse pregnancy-related outcomes to a health care provider.

17. The method of claim 16, wherein said communicating informs a subsequent treatment decision for said pregnant human patient and / or a neonate carried by said pregnant human patient.

18. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for preterm birth and a higher risk score for preterm birth, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of aspirin, a progesterone treatment, tocolytics to delay labor, transvaginal cervical length screenings, weekly nurse telephonic support, bed rest, or reduced physical activity.

19. The method of claim 18, wherein said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-a hydroxyprogesterone caproate, or vaginal progesterone.

20. The method of claim 19, wherein said 17-a hydroxyprogesterone caproate is administered as an injection.

21. The method of claim 19, wherein said vaginal progesterone is administered in gel form.

22. The method of claim 18, wherein the preventive intervention further comprises transvaginal cervical length screenings.78ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT23. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for preeclampsia and a higher risk score for preeclampsia, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of magnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity and bed rest.

24. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for GAB being less than 37 weeks and a higher risk score for GAB being less than 37 weeks, (i) the treatment decision for the pregnant human patient comprises a preventive intervention comprising administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity, and / or (ii) the treatment decision for the neonate carried by said pregnant human patient comprises a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU).

25. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for GDM and a higher risk score for GDM, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of insulin, an oral hypoglycemic agent, medical nutrition therapy, increased physical activity, and weight management.

26. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for gHTN and a higher risk score for gHTN, the treatment decision for said pregnant human patient comprises a preventive intervention comprising one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication, fetal monitoring, hospitalization, and bedrest.

27. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for low birthweight and a higher risk score for low birthweight, the treatment decision for the neonate comprises a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood79ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCToxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU).

28. The method of claim 17, wherein, when the plurality of adverse pregnancy-related outcomes comprises risk for RDS and a higher risk score for RDS, the treatment decision for said neonate comprises a preventive intervention comprising one or more of surfactant therapy, oxygen therapy, continuous airway pressure, mechanical ventilation, nutritional support, and prone positioning.

29. The method of any one of claims 1-9, wherein said measuring comprises contacting a capture agent that binds to IBP4 or SHBG to the biological sample.

30. The method of claim 29, wherein said capture agent is selected from the group consisting of an antibody, antibody fragment, nucleic acid-based protein binding reagent, small molecule or variant thereof.

31. The method of claim 29, wherein said contacting is part of an assay selected from the group consisting of enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).

32. The method of claim 29, wherein said measuring further comprises mass spectrometry (MS).

33. The method of claim 32, wherein said MS is co-immunoprecipitation-mass spectrometry (co-IP MS).

34. The method of any one of claims 1-33, wherein:a) the isolated biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from the group consisting of QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYI IPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID NO: 4), and / orb) the isolated biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from the group consisting of IALGGLLFPASNLR (SEQ ID NO: 5), GEDSSTSFCLNGLWAQGQR (SEQ80ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11).

35. A method of determining the probability of a plurality of adverse pregnancy-related outcomes, wherein the method comprises:(i) measuring in a biological sample obtained from a pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating a risk score using a reversal value for IBP4 and SHBG,wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the risk score is connected to the plurality of adverse pregnancy-related outcomes occurring, andwherein the risk score indicates an increased probability of two or more of the adverse pregnancy-related outcomes when the risk score is at or over 15% compared to a reference risk score, wherein the reference risk score is an algorithm-based biomarker- scored preterm birth risk.

36. The method of claim 35, wherein said plurality of adverse pregnancy-related outcomes comprises at least two or more of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

37. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising:81ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and(iii) administering to said pregnant human patient, wherein the pregnant human patient has a higher risk score in compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of aspirin, a progesterone treatment, tocolytics to delay labor, transvaginal cervical length screenings, weekly nurse telephonic support, bed rest, and / or reduced physical activity,wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy -related outcomes comprises preterm birth (PTB) and one or more adverse pregnancy -related outcomes selected from the group consisting of preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

38. The method of claim 37, wherein said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-a hydroxyprogesterone caproate, or vaginal progesterone.

39. The method of claim 38, wherein said 17-a hydroxyprogesterone caproate is administered as an injection.

40. The method of claim 38, wherein said vaginal progesterone is administered in gel form.

41. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising:82ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and(iii) administering to said pregnant human patient, wherein the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of magnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity and / or bed rest,wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy-related outcomes comprises preeclampsia (PE) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

42. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient and / or a neonate carried by said pregnant human patient, the method comprising:(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and83ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT(iii) where if the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, the method comprises: (a) administering to said pregnant human patient a preventive intervention comprising administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity, and / or (b) administering to the neonate a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy-related outcomes comprises predicted gestational age at birth (GAB) being less than 37 weeks and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

43. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising:(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and(iii) administering to said pregnant human patient, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of insulin, an oral hypoglycemic agent, medical nutrition therapy, increased physical activity, and weight management,84ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTwherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy-related outcomes comprises gestational diabetes mellitus (GDM) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

44. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient, the method comprising:(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and(iii) administering to said pregnant human patient, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication, fetal monitoring, hospitalization, and bedrest,wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy-related outcomes comprises gestational hypertension (gHTN) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), early (non-elective)85ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTcesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

45. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient, the method comprising:(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and(iii) administering to said neonate, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy-related outcomes comprises low birthweight and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and respiratory distress syndrome (RDS).

46. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient, the method comprising:86ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG);(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG); and(iii) administering to said neonate, where the pregnant human patient has a higher risk score compared to a reference risk score, or a plurality of higher risk scores than a corresponding plurality of reference risk scores, a preventive intervention comprising one or more of surfactant therapy, oxygen therapy, continuous airway pressure, mechanical ventilation, nutritional support, and prone positioning,wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring, andwherein the plurality of adverse pregnancy-related outcomes comprises respiratory distress syndrome (RDS) in the neonate and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and low birthweight.

47. The method of any one of claims 37-46, wherein the method further comprises combining said reversal value with one of more clinical factors selected from the group consisting of age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy to calculate said one or more risk scores for the plurality of adverse pregnancy-related outcomes occurring.

48. The method of any one of claims 37-47, further comprising an initial step of identifying said pregnant human patient as potentially at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium.87ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT49. The method of claim 48, wherein said one or more risk indicium is selected from the group consisting of age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections.

50. The method of claim 49, wherein said one or more risk indicium comprises BMI.

51. The method of claim 50, wherein said BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2.

52. The method of any one of claims 37-51, wherein said biological sample is selected from the group consisting of whole blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine.

53. The method of claim 52, wherein said biological sample is whole blood, plasma or serum.

54. The method of any one of claims 37-53, wherein said biological sample is obtained between 19 and 21 weeks of estimated gestational age.

55. The method of any one of claims 37-54, wherein the measuring comprises subjecting the biological sample to a proteomics workflow, wherein said proteomics workflow comprises quantification of a stable isotope labeled (SIS) surrogate peptide of said isolated biomarkers.

56. The method of any one of claims 37-55, wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS), wherein said MS is selected from the group consisting of:a) matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS);88ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTb) matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD));c) matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDI- TOF / TOF);d) surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS);e) electrospray ionization mass spectrometry (ESI-MS);f) electrospray ionization-mass spectrometry / mass spectrometry (ESI-MS / MS); g) electrospray ionization mass spectrometry / mass spectrometry^ (ESI- MS / (MS)n), wherein n is an integer greater than zero ;h) electrospray ionization 3D (ESI 3D) or linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS;i) i) electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS);j) electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); k) electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems);l) desorption / ionization on silicon (DIOS);m) secondary ion mass spectrometry (SIMS);n) atmospheric pressure chemical ionization mass spectrometry (APCI-MS); o) atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCI-MS / MS);p) atmospheric pressure chemical ionization-mass spectrometry^ (APCI-(MS)n) , wherein n is an integer greater than zero;q) ion mobility spectrometry (IMS);r) inductively coupled plasma mass spectrometry (ICP-MS);s) atmospheric pressure photoionization mass spectrometry (APPI-MS);t) atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPI-MS / MS); andu) atmospheric pressure photoionization-mass spectrometry^ (APPI-(MS)n), wherein n is an integer greater than zero.89ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT57. The method of any one of claims 37-55, wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS), wherein said MS comprises co-immunoprecipitation-mass spectrometry (co-IP MS).

58. The method of any one of claims 37-55, wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS), wherein said MS comprises liquid chromatography-mass spectrometry (LC-MS).

59. The method of any one of claims 37-55, wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS), wherein said MS comprises multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

60. The method of any one of claims 37-59, wherein:a) the isolated biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from the group consisting of QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYI IPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID NO: 4), and / orb) the isolated biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from the group consisting of IALGGLLFPASNLR (SEQ ID NO: 5), GEDSSTSFCLNGLWAQGQR (SEQ ID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11).

61. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of aspirin, a progesterone treatment, tocolytics to delay labor, transvaginal cervical length screenings, weekly nurse telephonic support, bed rest, and / or reduced physical activity,90ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTwherein said pregnant human patient has been identified for treatment by(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, andwherein the plurality of adverse pregnancy -related outcomes comprises preterm birth (PTB) and one or more adverse pregnancy -related outcomes selected from the group consisting of preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

62. The method of claim 61, wherein said preventive intervention comprising administration of a progesterone treatment comprises administration of progestogen, 17-a hydroxyprogesterone caproate, or vaginal progesterone.

63. The method of claim 62, wherein said 17-a hydroxyprogesterone caproate is administered as an injection.

64. The method of claim 62, wherein said vaginal progesterone is administered in gel form.

65. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of91ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTmagnesium sulfate, administration of low-dose aspirin, administration of antihypertensive medications, induction of labor, hospitalization, reduced physical activity, and bed rest,wherein said pregnant human patient has been identified for treatment by(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, andwherein the plurality of adverse pregnancy-related outcomes comprises preeclampsia (PE) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

66. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient and / or a neonate carried by said pregnant human patient by (a) administering to said pregnant human patient a preventive intervention comprising administration of antenatal corticosteroids to the pregnant human patient to accelerate fetal lung maturity, and / or (b) administering to said neonate a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, and a stay in a neonatal intensive care unit (NICU),wherein said pregnant human patient has been identified for treatment by92ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, andwherein the plurality of adverse pregnancy-related outcomes comprises predicted gestational age at birth (GAB) being less than 37 weeks and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

67. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of insulin, an oral hypoglycemic agent, medical nutrition therapy, increased physical activity, and weight management,wherein said pregnant human patient has been identified for treatment by(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),93ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTwherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, andwherein the plurality of adverse pregnancy-related outcomes comprises gestational diabetes mellitus (GDM) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

68. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a pregnant human patient by administering to said pregnant human patient a preventive intervention comprising one or more of administration of magnesium sulfate, administration of corticosteroids, administration of an antihypertensive medication, fetal monitoring, hospitalization, and bedrest,wherein said pregnant human patient has been identified for treatment by(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, and94ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTwherein the plurality of adverse pregnancy-related outcomes comprises gestational hypertension (gHTN) and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), early (non-elective) cesarean section (C-section), neonatal morbidity, low birthweight, and respiratory distress syndrome (RDS).

69. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient by administering to said neonate a preventive intervention comprising one or more of use of a temperature-controlled bed or incubator, tube feeding, monitoring of blood oxygen levels, monitoring of blood sugar levels, or a stay in a neonatal intensive care unit (NICU),wherein said pregnant human patient has been identified for treatment by(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, andwherein the plurality of adverse pregnancy-related outcomes comprises low birthweight and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and respiratory distress syndrome (RDS).95ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT70. A method for providing preventive intervention of one or more adverse pregnancy-related outcomes in a neonate carried by a pregnant human patient by administering to said neonate a preventive intervention comprising one or more of surfactant therapy, oxygen therapy, continuous airway pressure, mechanical ventilation, nutritional support, or prone positioning,wherein said pregnant human patient has been identified for treatment by(i) measuring in a biological sample obtained from said pregnant human patient levels of isolated biomarkers comprising Insulin Like Growth Factor Binding Protein 4 (IBP4) and Sex Hormone Binding Globulin (SHBG); and(ii) calculating one or more risk scores using a reversal value for IBP4 / SHBG, wherein the reversal value is based on a ratio of the level of said IBP4 over the level of said SHBG (IBP4 / SHBG),wherein the one or more risk scores are connected to a plurality of adverse pregnancy-related outcomes occurring,wherein said pregnant human patient is identified for treatment when said one or more risk scores are higher than a reference risk score, or a plurality of risk scores is higher than a corresponding plurality of reference risk scores, andwherein the plurality of adverse pregnancy-related outcomes comprises respiratory distress syndrome (RDS) in the neonate and one or more adverse pregnancy-related outcomes selected from the group consisting of preterm birth (PTB), preeclampsia (PE), predicted gestational age at birth (GAB) being less than 37 weeks, gestational diabetes mellitus (GDM), gestational hypertension (gHTN), early (non-elective) cesarean section (C-section), neonatal morbidity, and low birthweight.

71. The method of any one of claims 61-70, wherein:a) the isolated biomarker for IBP4 comprises a peptide fragment of IBP4 comprising an amino acid sequence selected from the group consisting of QCHPALDGQR (SEQ ID NO: 1), LPGGLEPK (SEQ ID NO: 2), THEDLYI IPIPNCDR (SEQ ID NO: 3), and EDARPVPQGSCQSELHR (SEQ ID NO: 4), and / or96ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTb) the isolated biomarker for SHBG comprises a peptide fragment of SHBG comprising an amino acid sequence selected from the group consisting of IALGGLLFPASNLR (SEQ ID NO: 5), GEDSSTSFCLNGLWAQGQR (SEQ ID NO: 6), DDWFMLGLR (SEQ ID NO: 7), SCDVESNPGIFLPPGTQAEFNLR (SEQ ID NO: 8), TWDPEGVIFYGDTNPK (SEQ ID NO: 9), VVLSSGSGPGLDLPLVLGLPLQLK (SEQ ID NO: 10), and ALALPPLGLAPLLNLWAKPQGR (SEQ ID NO: 11).

72. The method of any one of claims 61-71, wherein the method further comprises combining said reversal value with one of more clinical factors selected from the group consisting of age, BMI, medical history, race, hypertension / preeclampsia in a prior pregnancy, preterm birth in a prior pregnancy, cesarean section (C-section) delivery in a prior pregnancy, education level, first time mother, chronic diabetes, gestational diabetes in the current pregnancy, or hypertension or preeclampsia in the current pregnancy to calculate said one or more risk scores for the plurality of adverse pregnancy-related outcomes occurring.

73. The method of any one of claims 61-72, further comprising an initial step of identifying said pregnant human patient as potentially at risk of one or more adverse pregnancy-related outcomes based on one or more risk indicium.

74. The method of claim 73, wherein said one or more risk indicium is selected from the group consisting of age, prior pregnancy, history of previous low birth weight or preterm delivery, multiple 2nd trimester spontaneous abortion, prior first trimester induced abortion, familial and intergenerational factors, history of infertility, nulliparity, placental abnormalities, cervical and uterine anomalies, gestational bleeding, intrauterine growth restriction, in utero diethylstilbestrol exposure, multiple gestations, infant sex, short stature, low pre-pregnancy, weight / low body mass index (BMI), gravidity, fetal gender, diabetes, hypertension, hypothyroidism, asthma, education level, tobacco use, and urogenital infections.

75. The method of claim 74, wherein said one or more risk indicium comprises BMI.

76. The method of claim 75, wherein said BMI is greater than 22 kg / m2and less than or equal to 37 kg / m2.97ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCT77. The method of any one of claims 61-76, wherein said biological sample is selected from the group consisting of whole blood, plasma, serum, amniotic fluid, vaginal secretions, saliva, and urine.

78. The method of claim 77, wherein said biological sample is whole blood, plasma or serum.

79. The method of any one of claims 61-78, wherein said biological sample is obtained between 19 and 21 weeks of estimated gestational age.

80. The method of any one of claims 61-79, wherein measuring comprises subjecting the biological sample to a proteomics workflow comprising mass spectrometry (MS) or immunoassay.

81. The method of claim 80, wherein said proteomics workflow comprises quantification of a stable isotope labeled (SIS) surrogate peptide of said isolated biomarkers.

82. The method of claim 80, wherein said MS is selected from the group consisting of:a) matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS);b) matrix-assisted laser desorption / ionization time-of-flight post-source-decay (MALDI-TOF post-source-decay (PSD));c) matrix-assisted laser desorption / ionization time-of-flight / time-of-flight (MALDL TOF / TOF);d) surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS);e) electrospray ionization mass spectrometry (ESI-MS);f) electrospray ionization-mass spectrometry / mass spectrometry (ESI-MS / MS); g) electrospray ionization mass spectrometry / mass spectrometry^ (ESI-MS / (MS)n, wherein n is an integer greater than zero;h) electrospray ionization 3D (ESI 3D) or linear 2D ion trap mass spectrometry (ESI linear (2D)) ion trap MS;i) electrospray ionization triple quadrupole mass spectrometry (ESI triple quadrupole MS);98ACTIVE 718780158v1Attorney Docket No. 203123-030002 / PCTj) electrospray ionization quadrupole orthogonal time-of-flight (ESI Q-TOF); k) electrospray ionization Fourier transform mass spectrometry systems (ESI Fourier transform MS systems);l) desorption / ionization on silicon (DIOS);m) secondary ion mass spectrometry (SIMS);n) atmospheric pressure chemical ionization mass spectrometry (APCI-MS); o) atmospheric pressure chemical ionization-mass spectrometry / mass spectrometry (APCI-MS / MS);p) atmospheric pressure chemical ionization-mass spectrometry^ (APCI-(MS)n), wherein n is an integer greater than zero;q) ion mobility spectrometry (IMS);r) inductively coupled plasma mass spectrometry (ICP-MS);s) atmospheric pressure photoionization mass spectrometry (APPI-MS); t) atmospheric pressure photoionization-mass spectrometry / mass spectrometry (APPI-MS / MS); andu) atmospheric pressure photoionization-mass spectrometry^ (APPI-(MS)n), wherein n is an integer greater than zero.

83. The method of claim 80, wherein said MS comprises co-immunoprecipitationmass spectrometry (co-IP MS).

84. The method of claim 80, wherein said MS comprises liquid chromatographymass spectrometry (LC-MS).

85. The method of claim 80, wherein said MS comprises multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).99ACTIVE 718780158v1

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