Pregnancy biomarker profiles, methods and compositions related thereto

Abstract

The present invention provides methods and compositions related to biomarker profiles for each trimester of pregnancy. The present invention also provides methods for identifying patients at risk of developing a complication of pregnancy, such as preeclampsia. In further embodiments, the present invention relates to methods for the diagnosis of patients with preeclampsia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a cytokine array.

FIGS. 2A-2C are bar graphs depicting the serum levels as assessed by cytokine array of the indicated proteins in the first, second, and third trimesters of a normal pregnancy.

FIGS. 3A-3C are bar graphs depicting the serum levels as assessed by cytokine array of the indicated proteins in the first trimester of a normal pregnancy and pregnancies destined to be complicated by preeclampsia (“prediseased” pregnancies).

FIGS. 4A-4B are bar graphs depicting the serum levels as assessed by cytokine array of the indicated proteins in the first trimester of normal pregnancy and prediseased pregnancies.

FIG. 5 is a bar graph depicting the serum levels as assessed by Luminex® assays of the indicated proteins in the first trimester of normal pregnancies and in women who are not pregnant.

FIG. 6 is a bar graph depicting the serum levels as assessed by Luminex® assays of the indicated proteins in the first, second, and third trimesters of a normal pregnancy.

FIG. 7 is a bar graph depicting the serum levels as assessed by Luminex® assays of G-CSF in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 8 is a bar graph depicting the serum levels as assessed by Luminex® assays of IL-8 in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 9 is a bar graph depicting the serum levels as assessed by Luminex® assays of MCP-1 in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 10 is a bar graph depicting the serum levels as assessed by Luminex® assays of VEGF in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 11 is a bar graph depicting the serum levels as assessed by Luminex® assays of EGF in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 12 is a bar graph depicting the serum levels as assessed by Luminex® assays of Leptin in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 13 is a bar graph depicting secreted FasL levels as assessed by Luminex® assays in the first, second, and third trimesters of normal and prediseased pregnancies.

FIG. 14 is a bar graph depicting the serum levels as assessed by Luminex® assays of the indicated proteins in the first trimester of normal and prediseased pregnancies.

FIGS. 15A-15C are bar graphs depicting the serum levels as assessed by Luminex® assays of the indicated proteins in the first trimester of normal and prediseased pregnancies.

FIG. 16 is a graph depicting the effect of normal and preeclamptic serum on trophoblast cell viability.

FIG. 17 is a graph depicting the effect of serum from preeclamptic patients on Fas-mediated apoptosis.

FIG. 18 is a table depicting the patient characteristics relating to the data presented in FIGS. 18-20.

FIG. 19 is a graph depicting the effect of normal and pre-preeclamptic serum on trophoblast cell viability.

FIG. 20 is a graph depicting the effect of sera from first, second, and third trimester pregnancies on trophoblast cell viability.

FIG. 21 is a blot depicting the effect of preeclamptic sera on caspase-3 activation.

Claims

1. A method for determining or aiding in the determination that a pregnant woman is at risk of developing preeclampsia, comprising comparing the expression of one or more biomarkers in a blood sample from the pregnant woman to be assessed for risk of developing preeclampsia to a predetermined standard for each of the one or more biomarkers, wherein a significant difference in expression of the one or more biomarkers in the sample as compared to a predetermined standard of each of the one or more biomarkers indicates that the pregnant woman is at risk of developing preeclampsia, thereby determining or aiding in the determination that the pregnant woman is at risk of developing preeclampsia.

2. The method of claim 1, wherein the one or more biomarkers are selected from the group consisting of: IFNg, I-309, GM-CSF, GDNF, GCP-2, Fraktalkine, Flt-3 Ligand, FGF-7, FGF-6, Eotaxin-3, Eotaxin-2, Eotaxin, EGF, CNTF, CK b 8-1, BMP-6, BMP-4, BLC, BDNF, ANG, MCP-1, LIGHT, Leptin, IL-7, IL-6, IL-5, IL-4, IL-3, IL-2, IL-1ra, IL-1b, IL-1a, IL-16, IL-15, IL-13, IL-10, IGF-1, IGFBP-4, IGFBP-2, IGFBP-1, TNFB, TNFA, TGF-B3, TGF-B1, TARC, SDF-1, SCF, RANTES, PDGF-BB, PARC,NT-3, NAP-2, MIP-3A, MIP-1D, MIG, MDC, M-CSF, MCP-4, MCP-3, MCP-2, Lymphotactin, I-TAC, IL-8, IL-6R, IL-1 Ra, IL-17, IL-12 P70, IL-12 P40, IL-11, IL-1R1, IL-1 R4/ST2, IGF-1 SR, IGFBP-6, IGFBP-3, ICAM-3, ICAM-1, HGF, HCC-4, GRO-A, GRO, VEGF-D, VEGF, uPAR, TRAIL R4, TRAIL R3, Thrombopoietin, TIMP-2, TIMP-1, TECK, sTNF RI, sTNF RII, SGP130, PIGF, Oncostatin M, Steoprotegin, NT-4, MSP-A, MIP-3B, MIP-1B, MIP-1A, MIF, Fas, FasL, and tissue factor.

3. The method of claim 2, wherein the one or more biomarkers are selected from the group consisting of: Ang, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, Fas, EGF, IGFBP 1, MCP 1, IL8, and FasL.

4. The method of claim 1, wherein the predetermined standard corresponds to the expression levels of the one or more biomarkers in a pregnant woman who is not at risk of developing preeclampsia.

5. The method of claim 4, in which the pregnant woman to be assessed for risk of developing preeclampsia is in the first trimester of pregnancy, wherein the predetermined standard corresponds to the expression levels of the one or more biomarkers in the first trimester of pregnancy.

7. The method of claim 4, in which the pregnant woman to be assessed for risk of developing preeclampsia is in the second trimester of pregnancy, wherein the predetermined standard corresponds to the expression levels of said one or more biomarkers in the second trimester of pregnancy.

9. The method of claim 4, in which the pregnant woman to be assessed for risk of developing preeclampsia is in the third trimester of pregnancy, wherein the predetermined standard corresponds to the expression levels of said one or more biomarkers in the third trimester of pregnancy.

11. The method of claim 1, wherein the method comprises comparing the expression of two or more biomarkers and the determination of risk of developing preeclampsia is based on a score-based classification method.

12. The method of claim 1, wherein the method comprises comparing the expression of two or more biomarkers, wherein the determination of risk of developing preeclampsia is made by comparing the expression profile of the two or more biomarkers to a predetermined standard profile for the biomarkers, and wherein a difference in the profiles determines or aids in the determination that a pregnant woman is at risk of developing preeclampsia.

13. The method of claim 12, wherein the predetermined standard profile corresponds to the expression profile of the two or more biomarkers in a pregnant woman who is not at risk of developing preeclampsia.

14. The method of claim 13, in which the pregnant woman to be assessed for risk of developing preeclampsia is in the first trimester of pregnancy, wherein the predetermined standard profile corresponds to the expression profile of said two or more biomarkers in the first trimester of pregnancy.

16. The method of claim 13, in which the pregnant woman to be assessed for risk of developing preeclampsia is in the second trimester of pregnancy, wherein the predetermined standard profile corresponds to the expression profile of said two or more biomarkers in the second trimester of pregnancy.

18. The method of claim 13, in which the pregnant woman to be assessed for risk of developing preeclampsia is in the third trimester of pregnancy, wherein the predetermined standard profile corresponds to the expression profile of said two or more biomarkers in the third trimester of pregnancy.

20. The method of claim 12, wherein the predetermined standard profile is determined by comparing the expression of the two or more biomarkers in a pregnant woman to be assessed for risk of developing preeclampsia to the expression of the two or more biomarkers in a pregnant woman who is not at risk of developing preeclampsia using a machine learning technique.

21. The method of claim 1, wherein the predetermined standard profile is determined by comparing the expression of the two or more biomarkers in the pregnant woman to be assessed for risk of developing preeclampsia to the expression of the two or more biomarkers in a pregnant woman that who is not at risk of developing preeclampsia using support vector machines, K-nearest neighbor classifier, or classification tree analysis.

22. The method of claim 1, wherein the one or more biomarkers is selected from the group consisting of: Ang, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, and Fas and an increase in the expression of the one or more biomarkers as compared to the predetermined standard indicates that the pregnant woman is at risk of developing preeclampsia.

23. The method of claim 1, wherein the one or more biomarkers is selected from the group consisting of: EGF, IGFBP 1, MCP 1, IL8, and FasL and a decrease in the expression of the one or more biomarkers as compared to the predetermined standard indicates that the pregnant woman is at risk of developing preeclampsia.

24. The method of claim 1, wherein the expression of the one or more biomarkers is detected using a reagent that detects the one or more biomarkers.

25. The method of claim 24, wherein the reagent is an antibody or fragment thereof that binds the biomarker.

26. The method of claim 25, wherein the reagent is directly or indirectly labeled with a detectable substance.

27. The method of claim 24, wherein the expression of the one or more biomarkers is detected using mass spectroscopy.

28. The method of claim 1, wherein the expression of the one or more biomarkers is detected by: (a) detecting the expression of a polypeptide which is regulated by the one or more biomarker; (b) detecting the expression of a polypeptide which regulates the biomarker; or (c) detecting the expression of a metabolite of the biomarker.

29. A kit for determining if a pregnant woman is at risk of developing preeclampsia, comprising: (a) a receptacle for receiving a sample;(b) one or more reagents for detecting one or more biomarkers selected from the group consisting of: Ang, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, Fas, EGF, IGFBP 1, MCP 1, IL8, and FasL;(c) a reference sample; and(d) instructions for use according to the method of claim 1.

30. The kit of claim 29, wherein the kit comprises one or more reagents for detecting two or more biomarkers.

31. A kit comprising one or more reagents for detecting two or more biomarkers selected from the group consisting of: IFNg, I-309, GM-CSF, GDNF, GCP-2, Fraktalkine, Flt-3 Ligand, FGF-7, FGF-6, Eotaxin-3, Eotaxin-2, Eotaxin, EGF, CNTF, CK b 8-1, BMP-6, BMP-4, BLC, BDNF, ANG, MCP-1, LIGHT, Leptin, IL-7, IL-6, IL-5, IL-4, IL-3, IL-2, IL-1ra, IL-1b, IL-1a, IL-16, IL-15, IL-13, IL-10, IGF-1, IGFBP-4, IGFBP-2, IGFBP-1, TNFB, TNFA, TGF-B3, TGF-B1, TARC, SDF-1, SCF, RANTES, PDGF-BB, PARC,NT-3, NAP-2, MIP-3A, MIP-1D, MIG, MDC, M-CSF, MCP-4, MCP-3, MCP-2, Lymphotactin, I-TAC, IL-8, IL-6R, IL-1 Ra, IL-17, IL-12 P70, IL-12 P40, IL-11, IL-1R1, IL-1 R4/ST2, IGF-1SR, IGFBP-6, IGFBP-3, ICAM-3, ICAM-1, HGF, HCC-4, GRO-A, GRO, VEGF-D, VEGF, uPAR, TRAIL R4, TRAIL R3, Thrombopoietin, TIMP-2, TIMP-1, TECK, sTNF RI, sTNF RII, SGP130, PIGF, Oncostatin M, Steoprotegin, NT-4, MSP-A, MIP-3B, MIP-1B, MIP-1A, MIF, Fas, FasL, and tissue factor.

32. The kit of claim 31, wherein the biomarkers are selected from the group consisting of: Ang, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, Fas, EGF, IGFBP 1, MCP 1, IL8, and FasL.

33. The kit of claim 31, wherein the biomarkers correspond to a protein profile specific for the first, second, and/or third trimester of pregnancy.

34. The kit of claim 33, wherein the biomarkers correspond to a protein profile specific for the first trimester of pregnancy.

35. The kit of claim 33, wherein the biomarkers correspond to a protein profile specific for the second trimester of pregnancy.

36. The kit of claim 33, wherein the biomarkers correspond to a protein profile specific for the third trimester of pregnancy.

37. The kit of claim 34, wherein the kit comprises at least three biomarkers selected from the group consisting of: ANG, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, Fas, EGF, IGFBP 1, MCP 1, IL8, and FasL.

38. The kit of claim 31, further comprising a reference sample.

39. A method of identifying a compound useful to treat preeclampsia, comprising: (a) identifying a candidate compound which regulates the expression of one or more biomarkers selected from the group consisting of: Ang, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, Fas, EGF, IGFBP 1, MCP 1, IL8, and FasL; and(b) determining whether such candidate compound is effective to treat preeclampsia,wherein if the candidate compound is effective to treat preeclampsia, a compound useful to treat preeclampsia is identified.

40. The method of claim 39, comprising identifying a candidate compound that regulates the expression of two or more biomarkers.

41. A method of identifying preeclampsia risk biomarkers, comprising: (a) identifying two or more biomarkers that are potentially associated with a risk of developing preeclampsia;(b) comparing the level of expression of the biomarkers identified in step (a) in a first population of pregnant women at risk of developing preeclampsia to the expression of said two or more biomarkers in pregnant women not at risk of developing preeclampsia;(c) selecting biomarkers exhibiting a significant difference in expression in the first population of pregnant women at risk of developing preeclampsia;(d) comparing the level of expression of the biomarkers identified in step (c) in a second population of pregnant women at risk of developing preeclampsia to the expression of the two or more biomarkers in pregnant women not at risk of developing preeclampsia; and(e) selecting biomarkers exhibiting a significant difference in expression in the second population of pregnant women at risk of developing preeclampsia,wherein the biomarkers identified in step (e) are preeclampsia risk biomarkers.

42. The method of claim 41, further comprising: (f) comparing the level of expression of the biomarkers identified in step (e) in a third population of pregnant women at risk of developing preeclampsia to the expression of the two or more biomarkers in pregnant women not at risk of developing preeclampsia, wherein the expression of the biomarkers is detected by using a different assay format than used in claim 41; and(g) selecting biomarkers exhibiting a significant different in expression in said third population of pregnant women at risk of developing preeclampsia,wherein the biomarkers identified in step (g) are candidate biomarkers for risk of developing preeclampsia.

43. The method of claim 41, further comprising determining whether the biomarkers identified in step (e) distinguish between a pregnant woman at risk of developing preeclampsia and a pregnant woman not at risk of developing preeclampsia in a blind study.

44. The method of claim 42, further comprising determining whether the biomarkers identified in step (g) distinguish between a pregnant woman at risk of developing preeclampsia and a pregnant woman not at risk of developing preeclampsia in a blind study.

45. The method of claim 1, further comprising: (a) culturing human trophoblast cells in the presence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia;(b) culturing an equivalent sample of human trophoblast cells under the same conditions as cells in (a) but in the absence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia; and(c) comparing viability of cells cultured in (a) with the viability of cells cultured in (b),wherein if fewer cells cultured in (a) than cells cultured in (b) are viable, the pregnant woman is determined to be at risk of developing preeclampsia.

46. The method of claim 1, further comprising: (a) culturing human trophoblast cells in the presence of (i) anti-Fas antibodies and (ii) serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia;(b) culturing an equivalent sample of human trophoblast cells under the same conditions as cells in (a) but in the absence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia; and(c) comparing viability of cells cultured in (a) with the viability of cells cultured in (b),wherein if fewer cells cultured in (a) than cells cultured in (b) are viable, the pregnant woman is determined to be at risk of developing preeclampsia.

47. The method of claim 1, further comprising: (a) culturing human trophoblast cells in the presence of anti-Fas antibodies;(b) culturing cells from (a) in the presence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia;(c) culturing an equivalent sample of cells from (a) under the same conditions as cells in (b) but in the absence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia; and(d) comparing viability of cells cultured in (b) with the viability of cells cultured in (c),wherein if fewer cells cultured in (b) than cells cultured in (c) are viable, the pregnant woman is determined to be at risk of developing preeclampsia.

48. The method of claim 1, further comprising: (a) culturing human trophoblast cells in the presence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia;(b) culturing an equivalent sample of human trophoblast cells under the same conditions as cells in (a) but in the absence of serum or plasma obtained from the pregnant woman to be assessed for risk of developing preeclampsia;(c) determining if cells cultured in (a) undergo apoptosis;(d) determining if cells cultured in (b) undergo apoptosis,wherein if more cells cultured in (a) undergo apoptosis than cells cultured in (b), the pregnant woman is determined to be at risk of developing preeclampsia.

49. The method of claim 48, further comprising: (e) culturing an equivalent sample of cells in (a) but in the presence of serum or plasma obtained from a normal control; and(f) determining if cells cultured in (e) undergo apoptosis,wherein if more cells cultured in (a) undergo apoptosis than cells cultured in (e) the pregnant woman is determined to be at risk of developing preeclampsia.

50. The method of claim 48, wherein apoptosis is determined in (c) and (d) by detecting an apoptotic marker.

51. The method of claim 50, wherein the apoptotic marker is active caspase-3.

52. The method of claim 51, wherein the active caspase-3 is selected from p17 and p19.

53. The method of claim 45, further comprising: (d) determining if cells cultured in (a) undergo apoptosis;(e) determining if cells cultured in (b) undergo apoptosis,wherein if more cells cultured in (a) undergo apoptosis than cells cultured in (b), the pregnant woman is determined to be at risk of developing preeclampsia.

54. A kit for determining if a pregnant woman is at risk of developing preeclampsia, comprising: (a) a receptacle for receiving a sample;(b) one or more reagents for detecting one or more biomarkers selected from the group consisting of: Ang, Leptin, RANTES, PDGF, ICAM 1, VEGF, G-CSF, Fas, EGF, IGFBP 1, MCP 1, IL8, and FasL;(c) a reference sample;(d) trophoblast cells;(e) media suitable for growth of trophoblast cells;(f) a container for culturing trophoblast cells; and(g) instructions for use according to the method of claim 48.

55. The kit of claim 54, wherein the trophoblast cells are H8 trophoblast cells.

56. The method of claim 45, further comprising: (d) culturing an equivalent sample of human trophoblast cells under the same conditions as cells in (a) but in the presence of serum or plasma obtained from a normal control; and(e) comparing viability of cells cultured in (a) with the viability of cells cultured in (d),wherein if fewer cells cultured in (a) than cells cultured in (d) are viable, the pregnant woman is at risk of developing preeclampsia.

57. The method of claim 46, further comprising: (d) culturing an equivalent sample of human trophoblast cells under the same conditions as cells in (a) but in the presence of serum or plasma obtained from a normal control; and(e) comparing viability of cells cultured in (a) with the viability of cells cultured in (d),wherein if fewer cells cultured in (a) than cells cultured in (d) are viable, the pregnant woman is at risk of developing preeclampsia.

58. The method of claim 47, further comprising: (e) culturing an equivalent sample of cells from (a) under the same conditions as cells in (b) but in the presence of serum or plasma obtained from a normal control; and(f) comparing viability of cells cultured in (b) with the viability of cells cultured in (e),wherein if fewer cells cultured in (b) than cells cultured in (e) are viable, the pregnant woman is at risk of developing preeclampsia.