Biomarker for predicting the risk for postpartum hemorrhage

Abstract

The present disclosure provides a biomarker and methods for use in diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) in a pregnant subject. The biomarker includes Platelet-Derived Growth Factor-BB (PDGF-BB). Also provided are compositions comprising the biomarker, methods of processing a biological sample obtained from a pregnant subject, kits for detecting PDGF-BB, methods of preventing PPH, and methods of treating PPH.

Description

[0001] Docket No. 650053,01262

[0002] BIOMARKER FOR PREDICTING THE RISK FOR POSTPARTUM HEMORRHAGE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U. S. Provisional Application No. 63 / 747,723, filed January 21, 2025, U. S. Provisional Application No. 63 / 855,928, filed on August 1, 2025. U. S. Provisional Application No. 63 / 864,781, filed on August 15, 2025, and U. S. Provisional Application No. 63 / 903,586, filed on October 22, 2025, the contents of each of which are herein incorporated by reference in their entireties,

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number R61 HL 154254 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] BACKGROUND

[0007] Postpartum hemorrhage (PPH). or severe bleeding after childbirth, is the leading cause of maternal morbidity and mortality worldwide, affecting millions of women each year. While death from PPH is often preventable with prompt medical and surgical treatments, existing risk stratification tools only identify 60-85% of patients who experience PPH. In addition, no biomarkers of PPH are currently available which can allow for early risk prediction and / or diagnosis of PPH. Accordingly, there is an urgent need to better risk stratify and predict who will experience a PPH, while simultaneously developing novel treatment strategies for PPH.

[0008] SUMMARY

[0009] In an aspect, the disclosure provides a biomarker for use in diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) in a pregnant subject. The biomarker is Platelet-Derived Growth Factor-BB (PDGF-BB), and the PDGF-BB is present in the subject’s serum or plasma at or below a threshold concentration or a control concentration. The threshold concentration may be 11.5 pg / mL or 0.5 pg / mL.

[0010] In another aspect, the disclosure provides a method of sample processing. The method comprises producing a fraction of a biological sample obtained from a pregnant subject and quantifying an analyte within the fraction. Producing a fraction of the biological sample comprises introducing the biological sample to an antibody immobilized to a solid support, under conditions sufficient to bind the antibody to the analyte, and selectively removing components of the biological sample that are not bound to the antibody. The biological sample is a blood sample, a plasma sample, or a serum sample, and the analyte is PDGF-BB.Docket No. 650053,01262

[0011] In another aspect, the disclosure provides a method of treatment. The method comprises quantifying PDGF-BB in a biological sample obtained from a pregnant subject, and administering a treatment for postpartum hemorrhage when a quantity of the PDGF-BB is below a threshold concentration or a control concentration. The threshold concentration may be 0.5 pg / mL or 11.5 pg / mL, and the treatment for postpartum hemorrhage may comprise administering a uterotonic agent, administering a blood product, performing a tamponade procedure, performing controlled cord contraction and / or active management of the second stage of labor, performing a surgical procedure, and / or performing a uterine massage

[0012] In another aspect, the disclosure provides a kit for detecting PDGF-BB in a biological sample from a pregnant subject. The kit comprises a PDGF-BB-specific antibody immobilized to a bead or ELISA plate, and instructions for performing an ELISA assay. The kit may further comprise instructions for calculating a relative risk that the subject will develop postpartum hemorrhage.

[0013] In another aspect, the disclosure provides a method for diagnosing or predicting a risk of developing postpartum hemorrhage. The method comprises obtaining a plasma or serum sample from a pregnant subject during pregnancy or following childbirth, detecting PDGF-BB in the sample, and determining a concentration of PDGF-BB in the sample of about 11.5 pg / mL or less.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an illustration demonstrating the interaction between Platelet-Derived Growth Factor-BB (PDGF-BB), ciliary protein ARL 13B, and vascular stability protein PAK2. PDGF-BB induces ciliary signals, and ciliary signals in turn induce PDGF-BB signals, which creates an autocrine loop. PDGF-BB, and vascular endothelial growth factor-A (VEGF-A), trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor.

[0015] FIGS. 2A-2C depict ELISA (A) and western blot (B, C) results showing PDGF-BB levels in plasma samples from control subjects, subjects with postpartum hemorrhage (PPH), subjects with PPH and preeclampsia (PE), and subjects with PE. (A) Ihe ELISA results demonstrate that PDGF-BB levels are lower (<0.5 pg / mL) in patients who have PPH (n=23, P<0.001) as compared to those patients without PPH (~1 pg / mL, PE. n=41, PO.821 and Controls, n=10). (B, C) Western blot analysis also demonstrated a significant reduction in plasma PDGF-BB in subjects with PPH (P<0.001; n=3). The plasma concentrations of three additional ciliary markers (ARL13B, y-tubulin, IFT88) were also assessed.

[0016] FIGS. 3A-3C show results from the analysis of PDGF-BB levels in retrospective samples. (FIG. 3A) ELISA analysis was performed for control (n=l 0), PPH (n=23). PPH+PEDocket No. 650053,01262

[0017] (n=6), and PE (n=41) human plasma samples. Representative quantification was performed by RayBiotech’s ELISA analysis tool and template provided in the manufacturer's protocol. Data are represented as mean ± SD. with statistical significance calculated against control values. Kruskal-Wallis test was used to compare PDGF-BB among groups. Multiple comparison adjustment was done using Dunn’s test. (FIG. 3B) Western blot analysis was performed for control, PPH, PPH+PE, and PE human plasma samples. Band sizes indicate proteins at 27 kDa (PDGF-BB) and 45 kDa (P-actin). Blot probed for P-actin antibody were stripped and reprobed for PDGF-BB antibody. Representative quantification of protein expression between groups. Results were presented as mean ± SD. N=3 per group. ANOVA was performed. P values were calculated relative to control samples and corrected for multiple comparisons using Dunnett's test. (FIG. 3C) ROC analysis was performed and Youden's index was used to identify the threshold of PDGF-BB to discriminate PPH from Control. PGDF-BB of 11.515 was the selected cutoff. The figure was assembled and generated in Biorender or SPSS.

[0018] FIG.4 shows ROC analysis of samples collected during the 2ndtrimester of pregnancy (PPH vs Control). For every unit increase in PDGF-BB, the likelihood of developing PPH was decreased by 31.5%. odds ratio (OR) and 95% confidence interval (CI) 0,685, 0.510-0.921, P=0.012 (see. Table 5).

[0019] FIG. 5 shows ROC analysis of samples collected during the 3rdtrimester of pregnancy (PPH vs Control). For every unit increase in PDGF-BB, the likelihood of developing PPH was decreased by 23.2%. odds ratio (OR) and 95% confidence interval (CI) 0.768, 0.590-0.999, P=0.049 (see. Table 6).

[0020] DETAILED DESCRIPTION

[0021] This disclosure presents a biomarkers, compositions, and methods for use in diagnosing or predicting a risk of developing postpartum hemorrhage (PPH), as well as methods of preventing and / or treating PPH. Severe bleeding after childbirth, or PPH, affects millions of women each year. PPH is unpredictable, common, and the leading cause of maternal morbidity and mortality’ worldwide (1). PPH results in severe maternal morbidity in the United States by requiring blood transfusions to restore the blood loss, and maintain normal clotting abilities, and overall circulation for the patient (2). Unfortunately, rates of PPH are increasing in recent years (3, 4). Death from PPH is often preventable with prompt medical and surgical treatment. These treatments may include blood transfusions, medications to help the uterus contract, mechanical tamponade / pressure, or surgical interventions such as removal of the uterus for source control via a peripartum hysterectomy (5). Therefore, there is an urgent need to betterDocket No. 650053.01262

[0022] risk stratify and predict who will experience a PPH, while simultaneously developing novel strategies for preventing and / or treating PPH.

[0023] Current medical options are limited and not fully effective due to side effects, contraindications, costs, and constrained access in low-resource settings. Existing risk stratification tools identify 60-85% of patients who will experience a PPH, yet these scoring systems misclassify up to 40% of patients who will not have a PPH and 1% of patients who do have a PPH. Existing scoring systems rely on established risk factors for PPH such as abnormal placentation, infection in the uterus, and prolonged labor (6, 7) To date, early risk prediction and diagnosis of PPH is not possible as biomarkers related to PPH are not available. A biomarker that could predict PPH early would be extremely beneficial from a risk mitigation and clinical preparedness perspective, as this will initiate preemptive care. Additionally, depending on the biomarker and biological pathway that it influences, a biomarker may inform novel therapeutic strategies to treat PPH.

[0024] Platelet-Derived Growth Factor-BB (PDGF-BB) is a mitogenic growth factor that is found in circulation and produced by a variety of cell types including endothelial cells, fibroblasts, vascular smooth muscle cells, and placental cytotrophoblasts (10). The inventors’ previous work focused on cilia, a microtubule-based organelle that senses blood flow in blood vessels (8), reported that PDGF-BB triggers PAK2-ARL13B ciliogenesis and signaling, and ciliary signals in turn induce PDGF-BB signals, which creates an autocrine loop that promotes vascular integrity and prevents hemorrhage (9; FIG. 1). Disruption of both PDGF-BB and cilia in this autocrine loop are associated with vascular instability and hemorrhage (9).

[0025] The Examples demonstrate that plasma levels of PDGF-BB may be lower in patients with PPH. In Example 1, PDGF-BB levels were analyzed in plasma samples collected at the time of child delivery from patients with and without PPH. ELISA (FIG. 2A) and western blot (FIG. 2B) analysis demonstrated decreased levels of PDGF-BB levels are lower in patients who have a PPH (n=23) compared to those patients without PPH (Controls, n=10; Preeclampsia, n=41). Moreover, (1) a threshold concentration of 0.504 pg / mL PDGF-BB provided perfect separation between PPH subjects and control subjects, and (2) each 0.1 unit increase in PDGF-BB, decreased the odds of developing PPH by 72.2% In Example 4, PDGF- BB levels were analyzed in samples collected after 21 weeks, 0 days gestation but prior to delivery from subjects who, at child delivery': were diagnosed with PPH alone (PPH; n=23); were diagnosed with PPH and PE (PPH+PE; n=6); were diagnosed with PE alone (PE; n=41); or had no diagnosis of PPH or PE (Control; n=10). ELISA assay results showed that PDGF- BB levels were significantly lower in PPH, compared to controls and the PE alone group.Docket No. 650053,01262

[0026] Higher PDGF-BB levels were inversely associated with PPH risk [OR 0.62, 95% CI: 0.44-0.88; p=0.0068], and ROC analysis identified a PDGF-BB cutoff of 11.5 pg / mL with 100% sensitivity and specificity (FIG. 3C). These results demonstrate a clear association between higher PDGF-BB levels and lower risk of PPH and suggest that lower PDGF-BB levels may be a marker of a pathological condition predisposed to and / or indicating the presence of PPH.

[0027] Also provided are compositions comprising the biomarkers, kits for detecting PDGF-BB, methods of processing a biological sample obtained from a pregnant subject, methods of preventing PPH, and methods of treating PPH

[0028] Biomarkers

[0029] One aspect of the present disclosure provides a biomarker. The biomarker is Platelet- Derived Growth Factor-BB (PDGF-BB),

[0030] Another aspect is the use of the disclosed biomarker, or compositions comprising the biomarker, in diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) m a pregnant subject. The biomarker for use is PDGF-BB. As used herein, “postpartum hemorrhage” or “PPH” refers to a condition characterized by severe bleeding / blood loss following childbirth. During a hemorrhage, blood escapes from the circulatory system from damaged blood vessels and compromised and permeable endothelium. Criteria for categorizing and / or classifying PPH are known in the art. In some embodiments, PPH is classified based on a cumulative blood loss greater than or equal to 1,000 mL. or blood loss accompanied by signs or symptoms of hypovolemia within 24 hours after the birth process (includes intrapartum loss) regardless of route of deliveiy. In some embodiments, the PPH is delayed PPH. As used herein, “delayed PPH” refers to PPH occurring between 24 hours to 6 weeks after delivery.

[0031] As used herein, a “subject” may be interchangeable with a “patient” or “individual” and means an animal, which may be a human or non-human animal, that may be in need of treatment. In embodiments, the subject is a pregnant human subject. A subject in need may be any subject that may have developed, or is at risk of developing, PPH.

[0032] As used herein, the term “marker” or “biomarker” refers to a biological molecule present in a subject at varying concentrations useful in predicting the risk or incidence of a disease or a condition. For example, the biomarker can be a protein. The biomarker, such as a protein biomarker, may be present in higher or lower amounts in a subject having or at risk of developing PPH. Provided herein is a Platelet-Derived Growth Factor-BB (PDGF-BB) biomarker. By way of example, but not by way of limitation, as described in the disclosed methods, the PDGF-BB biomarker can be used to detect, diagnose, or confirm diagnosis of PPH. or to predict a risk of developing PPH. In some embodiments, the PDGF-BB is presentDocket No. 650053.01262

[0033] in a sample from the subject, such as the subject's blood, serum or plasma. In embodiments, PDGF-BB in a sample, such as a subject’s blood, serum, or plasma, may be at a concentration of about 50 pg / mL or less, 25 pg / mL or less, 22.5 pg / mL or less, 20 pg / mL or less, 19 pg / mL or less, 18 pg / mL or less, 17 pg / mL or less, 16 pg / mL or less, 15 pg / mL or less, 14 pg / mL or less, 13 pg / mL or less, 12.5 pg / mL or less, 12 pg / mL or less, 11.5 pg / mL or less, 11 pg / mL or less, 10 pg / mL or less, 5 pg / mL or less, 2.5 pg / mL or less, 1 pg / mL or less, 0.75 pg / mL or less, 0.5 pg / mL or less, 0.4 pg / mL or less, 0.3 pg / mL or less. 0.2 pg / mL or less. 0.1 pg / mL or less, 0.075 pg / mL or less, 0.05 pg / mL or less, 0.025 pg / mL or less, 0.01 pg / mL or less, 0.0075 pg / mL or less, 0.005 pg / mL or less, 0.0025 pg / mL or less, 0.001 pg / mL or less, 0.0005 pg / mL or less, or 0.0001 pg / mL or less, or is present at a concentration within a range bounded by any of the foregoing. As demonstrated in the Examples, the inventors discovered that the threshold concentration of PDGF-BB that indicates the presence of or risk of developing PPH was distinct for samples obtained during pregnancy and samples obtained immediately following childbirth. In samples obtained between weeks 21 to 40 of pregnancy, a threshold concentration of 11.5 pg / mL in plasma predicted a risk of developing PPH with 100% sensitivity and specificity. In samples obtained at the time of childbirth, a threshold concentration of 0.5 pg / mL in plasma predicted a risk of developing PPH with 100% sensitivity and specificity Thus, for samples obtained at different times during pregnancy or following childbirth, the concentration of PDGF-BB may be the same or may be independently selected. In some embodiments, the sample is collected during labor for childbirth or immediately following childbirth, and the threshold concentration is about 5 pg / mL, about 2.5 pg / mL, about 1 pg / mL, about 0.75 pg / mL, about 0.7 pg / mL, about 0.65 pg / mL, about 0.6 pg / mL, about 0.55 pg / mL, about 0.5 pg / mL, about 0.45 pg / mL, about 0.4 pg / mL, about 0.35 pg / mL, about 0.3 pg / mL, about 0.2 pg / mL, about 0.1 pg / mL, about 0.075 pg / mL, or about 0.05 pg / mL, or is within a range bounded by any of the foregoing In some embodiments, the sample is collected during the first, second or third trimester of pregnancy, and the threshold concentration is about 25 pg / mL, about 20 pg / mL, about 15 pg / mL, about 14 pg / mL, about 13 pg / mL, about 12.5 pg / mL, about 12 pg / mL, about 11.5 pg / mL, about 11 pg / mL, about 10.5 pg / mL, about 10 pg / mL, about 9 pg / mL, about 8 pg / mL, about 7 pg / mL, about 6 pg / mL. about 5 pg / mL, about 2.5 pg / mL, or about 1.5 pg / mL, or is within a range bounded by any of the foregoing.

[0034] In some embodiments, the level of PDGF-BB in the biological sample predicts a risk of developing PPH. In some embodiments, the biological sample is collected during the first trimester. In some embodiments, the biological sample is collected prior to week 14 of pregnancy. In some embodiments, the biological sample is collected during the secondDocket No. 650053.01262

[0035] trimester. In some embodiments, the biological sample is collected at a time point from week 14 to week 28 of pregnancy. In some embodiments, the biological sample is collected during the third trimester. In some embodiments, the biological sample is collected at a time point subsequent to week 28 of pregnancy.

[0036] Methods

[0037] Also provided in this disclosure is a method of sample processing. The method comprises obtaining a biological sample from a pregnant subject and producing a fraction of the biological sample. The term "‘biological sample” or “sample” as used herein includes, but is not limited to, a sample containing tissues, cells, and / or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and tears. In some embodiments, the biological sample is a biopsy (such as a tumor biopsy). A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third part)’ (e.g., received from an intermediary, such as a healthcare provider or lab technician). In the embodiments, the biological sample may be selected from the group consisting of a tissue sample, a cell sample, a biopsy sample, a blood sample, a lymph sample, a serum sample, a plasma sample, a urine sample, a saliva sample, a mucus sample, and a tear sample In some aspects, the biological sample is a blood sample, a plasma sample, or a serum sample. In some embodiments, the biological sample may be processed to reduce or prevent the measurement of platelet-released PDGF-BB. allowing for measurement of circulating PDGF-BB. For example, and without limitation, blood may be collected into CTAD (citrate-theophylline-adenosine-dipyridamole) tubes with optional platelet-activation inhibitors (e.g., prostaglandin El or apyrase) to suppress platelet activation and / or may be processed to generate a platelet-free plasma (PFP) sample (e.g., via two-step centrifugation, with a low-g spin followed by >10,000*g). Thus, in some embodiments, the biological sample is a PFP sample.

[0038] The sample may be obtained at any suitable time during pregnancy, during labor for or following childbirth. In some embodiments, the sample is obtained during the first trimester (e.g., weeks 1-13) of pregnancy, during the second trimester (e.g.. weeks 14-27) of pregnancy, during the third trimester (e.g., weeks 28-40) of pregnancy, during labor for childbirth, immediately following childbirth, and / or within 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours. 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after childbirth, or is obtained within a range bounded by any of the foregoing.

[0039] In an aspect, producing a fraction comprises introducing the biological sample to an antibody under conditions sufficient to bind the antibody to PDGF-BB in the biological sample;Docket No. 650053.01262

[0040] and selectively removing components of the biological sample that are not bound to the antibody. In some aspects, the antibody is immobilized to a support, such as a solid support. A skilled artisan will be aware of methods for selectively removing the unbound components from the sample, including, without limitation, washing the sample. Suitable antibodies to PDGF-BB can be found in the art, or made by using a laboratory animal to make suitable monoclonal antibodies. Such methods are well known in the art. Exemplary suitable antibodies can be found commercially. Primary antibodies used herein include PDGF-BB (Cat#NBP1-58279, Novus Biologicals), ARL13B (Cat#17711-1-AP. Proteintech), y-tubulin (Cat#GTX113286, GeneTex), IFT88 (Cat#PA5-18467, Thermofisher), and p-actin (Cat#4970P, Cell Signaling Technology). However, this disclosure is not limited by these antibodies, and any suitable antibodies that are specific and sensitive and capable of strongly binding to PDGF-BB (a ‘PDGF-BB-specific antibody”) can be used in the practice of tire disclosure described herein. The antibody may be immobilized to any suitable solid support, including, without limitation, an ELISA plate or a bead.

[0041] In an aspect, the method further comprises quantifying an analyte within the fraction. In some embodiments, the primary antibody that is capable of binding the marker of cilium may be conjugated, for example directly conjugated, to a detectable marker. In some embodiments, a secondary reagent or secondary antibody capable of binding the primary antibody can be used. The secondary antibody may be conjugated to the detectable marker. In some embodiments, a control protein is also used as a measure for quantification of the PDGF- BB. For example, the control protein may be tested at multiple amounts and a curve produced correlating the signal (e.g. brightness of a signal) to the amount of protein to correlate the amount of the PDGF-BB in the sample. Such detection and quantification techniques are well known in the art.

[0042] Suitable detectable markers are known in the art and include, for example, fluorescent proteins (green fluorescent proteins (GFP), red fluorescent protein (RFP, dsRed, etc.), yellow fluorescent protein (YFP) EBFP, ECFP, mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawbery, mCherry, mGrape, mRaspberry, mPlum, etc. and include all known in the art (for example, those described in Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues Dmitriy M. Chudakov, Mikhail V. Matz. Sergey Lukyanov, and Konstantin A. Lukyanov, Physiological Reviews 2010 90:3, 1103-1163, incorporated by reference in its entirety), reporter enzymes, ligand / substrate binding (bi otin / strepta vidin), and others known in the art.Docket No. 650053.01262

[0043] When using a reporter enzyme, detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. Suitable enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other suitable enzymes include, but are not limited to, p-galactosidase, acety lcholinesterase, and catalase. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-deteclion (spectrophotometer, fluorometer, or luminometer).

[0044] In some embodiments, the method of detecting and / or quantifying is detecting and / or quantifying using an enzyme-linked immunoassay (ELISA). ELISA techniques are readily known in the art. The ELISA may be a direct ELISA, an indirect ELISA, or a sandwich ELISA, among others. In some aspects, the direct or indirect immobilization of markers on a surface can be used. In other embodiments, the primary or secondary antibody may be immobilized on a suitable surface. In some embodiments, ELISA is a single molecule ELISA using antibody- coated beads.

[0045] A skilled artisan will readily identify other suitable methods for detecting and / or quantifying an analyte that are known in the art, including, without limitation, western blotting and flow cytometry. In some embodiments, the quantifying is performed using flow cytometry using beads in which the detectable marker is conjugated, for example, by binding of the PDGF-BB to an antibody conjugated to the bead, or other suitable methods known in the art. In some embodiments, the method used to perform the quantifying has a lower limit of quantification of about 50 pg / mL or less, 25 pg / mL or less. 15 pg / mL or less, 12.5 pg / mL or less, 12 pg / mL or less, 11.5 pg / mL or less, 11 pg / mL or less, 10 pg / mL or less, 5 pg / mL or less, 2.5 pg / mL or less, 1 pg / mL or less, 0.75 pg / mL or less, 0.5 pg / mL or less, 0.4 pg / mL or less, 0.3 pg / mL or less, 0.2 pg / mL or less, 0.1 pg / mL or less, 0.075 pg / mL or less, 0.05 pg / mL or less. 0.025 pg / mL or less, 0.01 pg / mL or less, 0.0075 pg / mL or less, 0.005 pg / mL or less, 0.0025 pg / mL or less, 0.001 pg / mL or less, 0.0005 pg / mL or less, or 0.0001 pg / mL or less, or has a lower limit of quantification within a range bounded by any of the foregoing.

[0046] In some embodiments, a quantity of the PDGF-BB resulting from the quantifying indicates the presence of or a risk of developing PPH. when the quantity is lower than a threshold concentration. In some embodiments, the threshold concentration is about 50 pg / mL or less, 25 pg / mL or less, 22.5 pg / mL or less, 20 pg / mL or less, 19 pg / mL or less, 17 pg / mL or less, 16 pg / mL or less, 15 pg / mL or less, 14 pg / mL or less, 13 pg / mL or less, 12.5 pg / mL or less, 12 pg / mL or less, 11.5 pg / mL or less, 11 pg / mL or less. 10 pg / mL or less, 5 pg / mL or less, 2.5 pg / mL or less, 1 pg / mL or less, 0.75 pg / mL or less, 0.5 pg / mL or less, 0.4 pg / mL or less,Docket No. 650053.01262

[0047] 0.3 pg / mL or less, 0.2 pg / mL or less, 0.1 pg / mL or less, 0.075 pg / 'mL or less, 0.05 pg / mL or less, 0.025 pg / mL or less. 0.01 pg / mL or less. 0.0075 pg / mL or less, 0.005 pg / mL or less, 0.0025 pg / mL or less, 0.001 pg / mL or less, 0.0005 pg / mL or less, or 0.0001 pg / mL or less, or is within a range bounded by any of the foregoing., In some embodiments, the threshold concentration is about 50 pg / mL, about 25 pg / mL, about 22.5 pg / mL, about 20 pg / mL, about 19 pg / mL, about 17 pg / mL, about 16 pg / mL, about 15 pg / mL, about 14 pg / mL, about 13 pg / mL, about 12.5 pg / mL, about 12 pg / mL. about 11.5 pg / mL. about 11 pg / mL, about 10 pg / mL, about 5 pg / mL, about 2.5 pg / mL, about 1 pg / mL, about 0.75 pg / mL, about 0.5 pg / mL, about 0.4 pg / mL, about 0.3 pg / mL, about 0.2 pg / mL, about 0.1 pg / 'mL, about 0.075 pg / mL, about 0.05 pg / mL, about 0.025 pg / mL, about 0.01 pg / mL, about 0.0075 pg / mL, about 0.005 pg / mL, about 0.0025 pg / mL. about 0.001 pg / mL, about 0.0005 pg / mL, or about 0.0001 pg / mL, or is within a range bounded by any of the foregoing.

[0048] For samples obtained at different times during pregnancy or following childbirth, the threshold concentration may be the same or may be independently selected. In some embodiments, the sample is collected during labor for childbirth or immediately following childbirth, and the threshold concentration is about 5 pg / mL, about 2.5 pg / mL, about 1 pg / mL. about 0.75 pg / mL, about 0.7 pg / mL, about 0.65 pg / mL, about 0.6 pg / mL, about 0.55 pg / mL, about 0.5 pg / mL, about 0.45 pg / mL, about 0.4 pg / mL, about 0.35 pg / mL, about 0.3 pg / mL, about 0.2 pg / mL, about 0.1 pg / mL, about 0.075 pg / mL, or about 0.05 pg / mL, or is within a range bounded by any of the foregoing. In some embodiments, the sample is collected during the first, second or third trimester of pregnancy, and the threshold concentration is about 25 pg / mL, about 20 pg / mL, about 15 pg / mL, about 14 pg / mL, about 13 pg / mL, about 12.5 pg / mL, about 12 pg / mL, about 11.5 pg / mL, about 11 pg / mL. about 10.5 pg / mL, about 10 pg / mL, about 9 pg / mL, about 8 pg / mL, about 7 pg / mL, about 6 pg / mL, about 5 pg / mL. about 2.5 pg / mL, or about 1.5 pg / mL. or is within a range bounded by any of the foregoing.

[0049] In some embodiments, a quantity of the PDGF-BB resulting from the quantify ing indicates the presence of or a risk of developing PPH, when the quantity is lower than a control concentration PDGF-BB concentration in one or more control samples. Any suitable control samples may be used For example, the one or more control samples may be obtained from one or more control subjects (e.g, one or more pregnant subjects) who are not experiencing PPH at the time the sample was obtained and do not subsequently develop PPH. In some cases, the one or more control samples are obtained from the subject at a different timepoint, including, without limitation, at an earlier timepoint during pregnancy. In some embodiments, the quantity of the PDGF-BB resulting from the quantifying indicates the presence of or a risk of developingDocket No. 650053.01262

[0050] PPH when the quantity is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, or at least 10-fold lower than the control concentration.

[0051] In some embodiments, the method further comprises detecting and / or quantifying one or more marker of cilium. As used tn this disclosure, a “marker of cilium’' refers to a protein that is expressed on cilia or expressed on ceils, such as endothelial cells or other cell types, that include cilia. Turbulent blood flow can lead to removal of cilia and associated proteins from the cells. The removed cilia and associated proteins can be detected on red blood cells, platelets, white blood cells, and in body fluids. This free cilia and the associated proteins can thus be used as biomarkers for altered flow in the blood, that is associated with vascular dysfunction and injury, and / or organ dysfunction and / or organ injury. Any suitable marker of cilium may be detected and / or quantified, including, without limitation, ARL13B, y-tubulin, and / or IFT88. In some embodiments, a quantity of the one or more marker of cilium resulting from the quantifying indicates the presence of preeclampsia, which is a risk factor for PPH.

[0052] Also disclosed are methods of treatment of PPH. Methods include quantifying PDGF-BB in a biological sample obtained from a pregnant subject and administering a treatment for PPH. In such methods, treatment for PPH is administered when a quantity of the PDGF-BB is lower than a threshold concentration. In some embodiments, the threshold concentration is about 50 pg / mL or less, 25 pg / mL or less, 22,5 pg / mL or less, 20 pg / mL or less, 19 pg / mL or less, 17 pg / mL or less, 16 pg / mL or less. 15 pg / mL or less, 14 pg / mL or less, 13 pg / mL or less, 12.5 pg / mL or less, 12 pg / mL or less, 11.5 pg / mL or less, 11 pg / mL or less. 10 pg / mL or less, 5 pg / mL or less, 2.5 pg / mL or less, 1 pg / mL or less, 0.75 pg / mL or less, 0.5 pg / mL or less, 0.4 pg / mL or less, 0.3 pg / mL or less, 0.2 pg / mL or less, 0.1 pg / mL or less, 0.075 pg / mL or less, 0.05 pg / mL or less, 0.025 pg / mL or less, 0.01 pg / mL or less, 0.0075 pg / mL or less, 0.005 pg / mL or less, 0.0025 pg / mL or less, 0.001 pg / mL or less, 0.0005 pg / mL or less, or 0.0001 pg / mL or less, or is about 50 pg / mL, about 25 pg / mL, about 22.5 pg / mL, about 20 pg / mL, about 19 pg / mL, about 18 pg / mL, about 17 pg / mL, about 16 pg / mL, about 15 pg / mL, about 14 pg / mL, about 13 pg / mL, about 12.5 pg / mL, about 12 pg / mL, about 11.5 pg / mL, about 11 pg / mL. about 10 pg / mL. about 5 pg / mL, about 2.5 pg / mL, about 1 pg / mL, about 0.75 pg / mL, about 0.5 pg / mL, about 0.4 pg / mL, about 0.3 pg / mL, about 0.2 pg / mL, about 0. 1 pg / mL, about 0.075 pg / mL, about 0.05 pg / mL, about 0.025 pg / mL, about 0.01 pg / mL, about 0.0075 pg / mL, about 0.005 pg / mL, about 0.0025 pg / mL, about 0.001 pg / mL, about 0.0005 pg / mL, or about 0.0001 pg / mL, or is within a range bounded by any of the foregoing. For samples obtained at different times during pregnancy or following childbirth, the threshold concentration may beDocket No. 650053.01262

[0053] the same or may be independently selected. In some embodiments, the sample is collected during labor for childbirth or immediately following childbirth, and the threshold concentration is about 5 pg / mL, about 2.5 pg / mL, about 1 pg / mL, about 0.75 pg / mL, about 0.7 pg / mL, about 0.65 pg / mL, about 0.6 pg / mL, about 0.55 pg / mL, about 0.5 pg / mL, about 0.45 pg / mL, about 0.4 pg / mL, about 0.35 pg / mL, about 0.3 pg / mL, about 0.2 pg / mL, about 0.1 pg / mL, about 0.075 pg / mL, or about 0.05 pg / mL, or within a range bounded by any of the foregoing. In some embodiments, the sample is collected during the first, second or third trimester of pregnancy, and the threshold concentration is about 25 pg / mL, about 20 pg / mL, about 15 pg / mL, about 14 pg / mL, about 13 pg / mL, about 12.5 pg / mL, about 12 pg / mL, about 11.5 pg / mL, about 11 pg / mL, about 10.5 pg / mL, about 10 pg / mL, about 9 pg / mL, about 8 pg / mL, about 7 pg / mL, about 6 pg / mL, about 5 pg / mL. about 2,5 pg / mL, or about 1.5 pg / mL, or is within a range bounded by any of the foregoing.

[0054] In some embodiments, the treatment for PPH is administered when the quantity of PDGF-BB is lower than a control concentration PDGF-BB concentration in one or more control samples. In some embodiments, the treatment for PPH is administered when the quantity is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2.5-fold, at least 3-fold, at least 3 5-fold, at least 4-fold, at least 45-fold, at least 5-fold, or at least 10-fold lower than the control concentration.

[0055] The quantifying may be performed using any suitable method known in the art, including those described herein. In some embodiments, the quantifying is performed using an enzyme-linked immunoassay (ELISA). In some embodiments, the ELISA is a single-molecule ELISA.

[0056] As used herein, a “treatment for postpartum hemorrhage” refers to any medical intervention performed to prevent the incidence of PPH and / or reduce the morbidity and mortality of PPH. Suitable treatments are known m the art (15). In some embodiments, the treatment comprises administering a uterotonic agent, including, without limitation, oxytocin, methylergonovine, 15-methyl prostaglandin F2a, and misoprostol. In some embodiments, the uterotonic agent is administered during labor for childbirth, with or soon after the deliver, of the anterior shoulder of the fetus. In some embodiments, the treatment comprises administering a blood product (e.g., whole blood, red blood cells, platelets, plasma, cryoprecipitated AHF, white blood cells, granulocytes). The blood products may be suitably administered to treat coagulopathy. In some embodiments, the treatment comprises performing a tamponade procedure with mechanical (e.g., Bakri balloon. Jada device) or suction devices. In some embodiments, the treatment comprises performing controlled cord contractions and / or activeDocket No. 650053,01262

[0057] management of the second stage of labor. As used herein, “controlled cord contractions” refers to manual clamping of the fetus’ umbilical cord while applying suprapubic counterpressure. In some embodiments, the treatment comprises performing a uterine massage following delivery of the placenta. In some embodiments, the treatment comprises performing a surgical procedure such as dilation and curettage, interventional radiology’ guided embolization, and / or peripartum hysterectomy.

[0058] Also disclosed are methods for diagnosing or predicting a risk of developing PPH One such method comprises obtaining a sample from a pregnant subject (for example a blood, plasma, or serum sample) at any7time during pregnancy or following childbirth, detecting PDGF-BB in the sample, and determining a concentration of the PDGF-BB in the sample. The concentration of PDGF-BB in the sample may be about 50 pg / mL or less. 25 pg / mL or less, 22.5 pg / mL or less. 20 pg / mL or less, 19 pg / mL or less, 17 pg / mL or less, 16 pg / mL or less, 15 pg / mL or less, 14 pg / mL or less, 13 pg / mL or less, 12.5 pg / mL or less, 12 pg / mL or less, 11.5 pg / mL or less, 11 pg / mL or less, 10 pg / mL or less, 5 pg / mL or less, 2.5 pg / mL or less, 1 pg / mL or less, 0.75 pg / mL or less, 0.5 pg / mL or less, 0.4 pg / mL or less, 0.3 pg / niL or less. 0.2 pg / mL or less. 0.1 pg / mL or less, 0.075 pg / mL or less, 0.05 pg / mL or less. 0.025 pg / mL or less, 0.01 pg / mL or less, 00075 pg / mL or less. 0.005 pg / mL or less, 0.0025 pg / mL or less, 0.001 pg / mL or less, 0.0005 pg / mL or less, or 0.0001 pg / mL or less, or may be within a range bounded by any of the foregoing. In some embodiments, the concentration is within a range of about 1 fg / mL to about 11.5 pg / mL. In some embodiments, the sample is collected during labor for childbirth or immediately following childbirth, and the concentration is about 5 pg / mL or less, about 2.5 pg / mL or less, about 1 pg / mL or less, about 0.75 pg / mL or less, about 0.7 pg / mL or less, about 0.65 pg / mL or less, about 0.6 pg / mL or less, about 0.55 pg / mL or less, about 0.5 pg / mL or less, about 0.45 pg / mL or less, about 0.4 pg / mL or less, about 0.35 pg / mL or less, about 0.3 pg / mL or less, about 0.2 pg / mL or less, about 0.1 pg / mL or less, about 0.075 pg / mL or less, or about 0.05 pg / mL or less, or is within a range bounded by7any of the foregoing In some embodiments, the sample is collected during the first, second or third trimester of pregnancy, and the concentration is about 25 pg / mL or less, about 20 pg / mL or less, about 15 pg / mL or less, about 14 pg / mL or less, about 13 pg / mL or less, about 12.5 pg / mL or less, about 12 pg / mL or less, about 11.5 pg / mL or less, about 11 pg / mL or less, about 10.5 pg / mL or less, about 10 pg / mL or less, about 9 pg / mL or less, about 8 pg / mL or less, about 7 pg / mL or less, about 6 pg / mL or less, about 5 pg / mL or less, about 2.5 pg / mL or less, or about 1.5 pg / mL or less, or is within a range bounded by any of the foregoing. The determining a concentration of the PDGF-BB may be performed using any suitable method known in the art, including thoseDocket No. 650053.01262

[0059] described herein. In some embodiments, the determining is performed using an enzyme-linked immunoassay (ELISA). In some embodiments, the ELISA is a single-molecule ELISA (e.g., the SIMOA Single Molecule Array). In some embodiments, the ELISA is an automated sandwich ELISA (e.g., the Ella Automated Immunoassay System). The sample may be obtained at any suitable time during pregnancy, during labor for childbirth, or following childbirth. In some embodiments, the method further comprises calculating a relative risk of developing PPH based on the concentration of the PDGF-BB in the sample. In the Examples, the inventors discovered that a 0.1 unit decrease in PDGF-BB concentration decreased the odds of developing PPH by 72.2%. In some embodiments, the calculation of the relative risk of developing PPH is based on the concentration of PDGF-BB and the concentration of one or more marker of cilium. In some embodiments, the concentration of the one or more marker of cilium indicates the presence of preeclampsia. In some embodiments, the marker of cilium comprises ARL13B, y-tubulin, and / or IFT88.

[0060] Kits

[0061] Also provided herein is a kit for detecting PDGF-BB in a biological sample obtained from a pregnant subject. The kit may comprise a PDGF-BB-specific antibody, and instructions for performing an ELISA assay. The antibody may be provided immobilized to a support, such as a bead or an ELISA plate. In some embodiments, the ELISA assay is a single-molecule ELISA. In some embodiments, the kit further comprises at least one secondary antibody with a detection label. The kits contemplated herein can contain one or more antibody that binds to PDGF-BB. The kit may be configured to detect PDGF-BB m any suitable biological sample. In some embodiments, the biological sample is a blood sample, a serum sample, or a plasma sample. In some embodiments, the kit further comprises instructions for calculating a relative risk that the subject will develop PPH. In some embodiments, the kits further comprise one or more antibody that binds to one or more marker of cilium. In some embodiments, the one or more marker of cilium is associated with preeclampsia. In some embodiments, the one or more marker of cilium comprises ARL13B, y-tubulin, and / or IFT88.

[0062] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that Mil be apparent to one of skill in the art in light of the disclosure tliat follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps.Docket No. 650053.01262

[0063] are not meant to be construed to indicate any specific stnictures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and ail examples, or exemplary' language (e.g., "such as") provided herein, is intended merely' to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising.” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of' those certain elements.

[0064] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a therapeutic” or “an antibody” should be interpreted to mean “one or more therapeutics” and “one or more antibodies.’’ respectively, unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.”

[0065] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It -will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” wih be understood to include the possibilities of “A” or " B" or “A and B.”

[0066] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%. it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%. etc., are expresslyDocket No. 650053,01262

[0067] enumerated in this specification. Ihese are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term. All percentages referring to amounts are by weight unless indicated otherwise.

[0068] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0069] 'fire following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0070] EXEMPLARY EMBODIMENTS

[0071] Embodiment 1. A biomarker for use in diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) in a pregnant subject, wherein the biomarker is Platelet-Denved Growth Factor-BB (PDGF-BB), and wherein the PDGF-BB is present in the subject’s serum or plasma at or below a threshold concentration or a control concentration, optionally wherein the threshold concentration is about 11.5 pg / mL or 0.5 pg / mL, Embodiment 2. A method of sample processing, comprising:

[0072] (a) producing a fraction of a biological sample obtained from a subject, comprising:

[0073] (i) introducing the biological sample to an antibody under conditions sufficient to bind the antibody to an analyte in the biological sample, wherein the antibody is immobilized to a solid support; and

[0074] (ii) selectively removing components of the biological sample that are not bound to the antibody; andDocket No. 650053.01262

[0075] (b) quantifying the analyte within the fraction,

[0076] wherein the biological sample is a blood sample, a serum sample, or a plasma sample, wherein the subject is pregnant, and wherein the analyte is Pl telet-Derived Growth Factor-BB (PDGF-BB).

[0077] Embodiment 3. The method of embodiment 2, comprising performing (a)-(b) using an enzyme-linked immunoassay (ELISA).

[0078] Embodiment 4. The method of embodiment 3, wherein the ELISA is a single¬ molecule ELISA.

[0079] Embodiment 5. The method of any one of embodiments 2-4, wherein a quantify of the analyte resulting from the quantifying in (b) indicates a presence of or a risk of developing postpartum hemorrhage, when the quantify is lower than a threshold concentration or a control concentration.

[0080] Embodiment 6. The method of any one of embodiments 2-5, wherein the biological sample is obtained during the first trimester of pregnancy, during the second trimester of pregnancy, during the third trimester of pregnancy, during labor for childbirth, and / or immediately following childbirth.

[0081] Embodiment 7. The method of embodiment 5 or 6, wherein the threshold concentration is about 0.5 pg / mL, optionally wherein the biological sample is obtained during labor for childbirth or immediately following childbirth

[0082] Embodiment 8. The method of embodiment 5 or 6. wherein the threshold concentration is about 11.5 pg / mL, optionally wherein the biological sample is obtained during the second or third trimester of pregnancy.

[0083] Embodiment 9. The method of any one of embodiments 2-8, wherein the method has a lower limit of quantification (LLOQ) for PDGF-BB, and wherein the LLOQ is less than about 0.5 pg / mL.

[0084] Embodiment 10. A method of treatment, comprising:

[0085] (a) quantifying Platelet-Derived Growth Factor-BB (PDGF-BB) in a biological sample obtained from a pregnant subj ect;

[0086] (b) administering a treatment for postpartum hemorrhage, when a quantity of the PDGF-BB is lower than a threshold concentration or a control concentration.

[0087] Embodiment 11. The method of embodiment 10, wherein the biological sample is obtained during the first trimester of pregnancy, during the second trimester of pregnancy, during the third trimester of pregnancy, during labor for childbirth, and / or immediately following childbirth.Docket No. 650053.01262

[0088] Embodiment 12. The method of embodiment 10 or 11, wherein the threshold concentration is about 0.5 pg / mL, optionally wherein the biological sample is obtained during labor for childbirth or immediately following childbirth.

[0089] Embodiment 13. The method of embodiment 10 or 11, wherein the threshold concentration is about 11.5 pg / mL, optionally wherein the biological sample is obtained during the second or third trimester of pregnancy.

[0090] Embodiment 14. The method of any one of embodiments 10-13, wherein the quantifying is performed using an enzyme-linked immunoassay (ELISA).

[0091] Embodiment 15. The method of embodiment 14. wherein the ELISA is a single¬ molecule ELISA.

[0092] Embodiment 16. The method of any one of embodiments 10-15, wherein the biological sample is a blood sample, a serum sample, or a plasma sample.

[0093] Embodiment 17. The method of any one of embodiments 10-16, wherein the treatment for postpartum hemorrhage comprises administering a uterotonic agent, administering a blood product, performing a tamponade procedure, performing controlled cord contraction and / or active management of the second stage of labor, performing a surgical procedure, and / or performing a uterine massage, optionally wherein the surgical procedure comprises dilation and curettage, interventional radiology guided embolization, and / or peripartum hysterectomy.

[0094] Embodiment 18. The method of embodiment 17, wherein the uterotonic agent is selected from the group consisting of oxytocin, methylergonovine, 15-methyl prostaglandin F2a, misoprostol, and combinations thereof.

[0095] Embodiment 19. A kit for detecting Platelet-Derived Growth Factor-BB (PDGF-BB) in a biological sample obtained from a pregnant subject, the kit comprising:

[0096] (a) a PDGF-BB-specific antibody, wherein the antibody is immobilized to a bead or ELISA plate; and

[0097] (b) instructions for performing an ELISA assay.

[0098] Embodiment 20. The kit of embodiment 19, wherein the biological sample is a blood sample, a serum sample, or a plasma sample.

[0099] Embodiment 21. The kit of embodiment 19 or 20, further comprising instructions for calculating a relative risk that the subject will develop postpartum hemorrhage.

[0100] Embodiment 22. A method for diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) comprising:Docket No. 650053,01262

[0101] (a) obtaining a plasma or serum sample from a pregnant subj ect at any time during pregnancy or following childbirth;

[0102] (b) detecting Platelet-Derived Growth Factor-BB (PDGF-BB) in the sample; and (c) determining a concentration of the PDGF-BB in the sample, wherein the concentration is about 11.5 pg / mL or less.

[0103] Embodiment 23. The method of embodiment 22, wherein the sample is obtained during the first trimester of pregnancy, during the second trimester of pregnancy, during the third trimester of pregnancy, during labor for childbirth, and / or immediately following childbirth

[0104] Embodiment 24. The method of embodiment 22 or 23, wherein the concentration is about 0.5 pg / mL or less, optionally wherein the sample is obtained during labor for childbirth or immediately following childbirth..

[0105] Embodiment 25. The method of any one of embodiments 22-24, wherein the concentration of PDGF-BB is within a range of about 1 fg / mL to about 11.5 pg / mL.

[0106] Embodiment 26, The method of any one of embodiments 22-25, wherein the quantifying is performed using an enzyme-linked immunoassay (ELISA).

[0107] Embodiment 27. The method of embodiment 26, wherein the ELISA is a single¬ molecule ELISA.

[0108] Embodiment 28. The method of any one of embodiments 2-18 or 22-27, further comprising detecting and / or quantifying one or more marker of cilium.

[0109] Embodiment 29. The method of embodiment 28, wherein the one or more marker of cilium comprises ARL13B, y-tubulm, and / or IFT88.

[0110] Embodiment 30. Ihe method of any one of embodiments 2-18 or 22-29, wherein the biological sample is a platelet-free plasma sample.

[0111] EXAMPLES

[0112] Example 1: Altered Platelet-Derived Growth Factor-BB in Postpartum Hemorrhage To investigate the potential use of Platelet-Derived Growth Factor-BB (PDGF-BB) as a biomarker for postpartum hemorrhage (PPH), PDGF-BB plasma levels were analy zed in patients with and without PPH. Plasma samples, which were collected at the time of fetal delivery, were obtained from the Medical College of Wisconsin IRB tissue bank, and analyzed by enzyme-linked immunoassay (ELISA; FIG. 2A) and western blot (FIG. 2B). For western blot analysis, plasma samples were diluted with IX RIPA buffer and quantified for total proteins. Then the samples were added with IX laemmli buffer (sample buffer) and boiled for 5 minutes at 95° C and then loaded to 4-20% gradient gel at 20 ug concentration. The gel wasDocket No. 650053,01262

[0113] transferred to the nitrocellulose (NC) membrane for antibody probing, and the NC membrane blot was probed for PDGF-BB. ARL13B, IFT88, gamma-tubulin, and beta-actin antibodies (PDGF-BB Cat#NBP 1-58279, Novus Biologicals; ARL13B Cat#17711-l-AP, Proteintech; y-tubulin Cat#GTX113286, GeneTex; IFT88 Cat#PA5-1 467, Thermofisher; and P-actin Cat#4970P, Cell Signaling Technology) with the respective secondary antibody and imaged in chemiluminescence for documentation. The blots were quantified with ImageJ software and the data was ploted in GraphPad Prism software. The PDGF-BB ELISA assay (Cat#ELH-PDGFBB-1, RayBiotech), was performed as per the manufacturer’s instruction (which is incorporated herein by reference in its entirety ).

[0114] The ELISA and western blot analysis demonstrated that PDGF-BB levels are lower (<0.5 pg / mL) in patients who have aPPH (n=23, PO. OOl) compared to those patients without aPPH (~1 pg / mL, Preeclampsia, n=41, P=0.821 and Controls, n=10). Interestingly, athreshold value of 0.504 pg / mL PDGF-BB provided a perfect separation, with PPH patients below the threshold concentration and control patients above the threshold concentration.

[0115] These results demonstrate a clear association between higher PDGF-BB levels and lower risk of PPH (odds ratio and 95% Cl. <0.001, <0.001-0.028, P=0.0066) in samples collected at time of delivery7when compared to control. Specifically, for each 0 1 unit increase in PDGF-BB, the odds of developing PPH will decrease by 72.2%. These exciting data suggest that the temporal relationship and normogram (normal levels) of PDGF-BB in pregnancy should be explored. Based on these results, and without wishing to be bound by a particular theory, it was expected that picogram or lower levels of PDGF-BB in early7stages of pregnancy and / or delivery will be predictive of PPH.

[0116] Example 2

[0117] To assess the temporal relationship and normogram (normal levels) of PDGF-BB in pregnancy. PDGF-BB protein levels will be measured in the plasma and serum of pregnant women at each trimester of pregnancy, during delivery7hospitalization and immediately' after childbirth. The PDGF-BB levels will be analyzed by ELISA quantification (Cat#lQH- PDGFBB-1, RayBiotech, sensitivity- 0.2 pg / mL) in pregnant women at each trimester of pregnancy, childbirth hospitalization and after childbirth to establish the normogram and risk prediction for PPH. Controls for each time point will be nulliparous pregnant women who do not have a PPH. Samples will be procured from MCW tissue bank. The retrospective blood samples collected at different periods of the pregnant w omen will be assessed for PDGF-BB levels in both plasma and serum. The data obtained will be compared with the established PPH patient PDGF-BB levels.Docket No. 650053.01262

[0118] Using the incidence of PPH 5% versus 0% controls, n=304 samples would be required (152 no PPH and 152 PPH) will provide 80% power to detect tins difference and alpha 0.05 allowing for 10% dropout and set as a standard for the PDGF-BB levels post-delivery. Data obtained from the patients will be plotted for the AUC curve to identify the sensitivity of the detection range. The patient data collection will include patient demographics, age, previous history' of PE or PPH, and history of any health-related concerns especially hypertension or obesity to identify the patient population at risk of developing PE or PPH during pregnancy. Without wishing to be bound by any particular theory, it is expected that PDGF-BB protein levels in plasma will be lower than in serum and will be distinct at each stage of pregnancy.

[0119] Example 3

[0120] Hie majority of previous studies assessed PDGF-BB levels using ELISA assay on serum (11, 12, 13, 14), and for the majority' of studies, nanogram amounts of PDGF-BB were found in these samples. In this disclosure, PDGF-BB levels were assessed from the blood plasma of patients who had a PPH, which was observed to be present in picogram levels (0.5 pg or less). Data show's a clear correlation between higher PDGF-BB levels and lower risk of PPH (odds ratio and 95% CI. <0.001, <0.001-0.028, P=0.0066). Methods that can detect at low’ picogram to femtogram levels may' assist in better stratifying risk factors associated with PPH. Assays with femtogram sensitivity' are possible with modern technologies. The assay will be optimized for detecting PDGF-BB in both plasma and serum. We also anticipate that during early trimesters in pregnancy. PDGF-BB levels will vary and having a test that can detect <0.5 pg / mL and have a greater detection range will assist in risk factor stratification. Therefore, an assay will be developed that will detect the protein in the range of 1 fg / mL (or less) to 0.5 pg / mL in patient blood or serum. One potential assay system is the SIMOA fluorescent bead technology from Quanterix. SIMOA is an ultrasensitive fluorescent-based ELISA assay that uses antibody coated beads, a fluorescent conjugated detection antibody and a digital readout. SIMO A offers up to 1,000-fold greater sensiti vity than standard ELIS A by isolating individual immunocomplexes on femtoliter-sized beads and detecting them digitally. Commercially available antibody pairs for PDGF-BB can be used to develop the SIMOA assay kit, and the assay can be run on the SIMOA machine (HD-X™ Automated Immunoassay Analyzer).

[0121] A PDGF-BB detection assay will be developed using the Ella, automated Simple Plex immunoassay system offers sensitivity7for 0.291 pg / mL and the rapid turnaround for results in 90 minutes compared to conventional ELISA which is about 4-5 hours. This is an automated sandwich ELISA with microfluidic cartridge containing three Glass Nano Reactors, which avoids cross reactivity' and interference with PDGF-BB related molecules.Docket No. 650053,01262

[0122] Example 4: PDGF-BB as a Potential Biomarker for Early Diagnosis of Postpartum Hemorrhage

[0123] Objective

[0124] Postpartum hemorrhage (PPH) remains a leading cause of maternal morbidity and mortality worldwide, with significant implications for women's health. The United States has the highest maternal mortality rate at 11%, increasing from 8 to 40 cases per 10,000 deliveries.16Despite advances in obstetric care, early detection of PPH remains a major challenge. Current risk stratifying tools identify 60-85% of patients who will experience a PPH, yet these scoring systems misclassify7up to 40% of patients who will not experience PPH and 1% of patients who experience a PPH.6There remains a need for reliable biomarkers to accurately predict and diagnosis PPH early, which could allow for timely intervention and better management of PPH, ultimately improving maternal outcomes. Platelet-derived growth factor (PDGF-BB), a key cytokine is well established to stabilize the embryonic blood vasculature.17The objective was to explore the potential of PDGF-BB as a diagnostic marker for PPH, which this study investigated.

[0125] Study Design

[0126] A retrospective analysis w7as conducted of 80 plasma samples collected after 21 weeks 0 days gestation but prior to delivery. Samples were selected based on diagnosis in tissue bank at delivery of PPH (n=23), PPH 'ith preeclampsia (PPH+PE; n=6), and PE alone (n=41 ). Controls (n=10) were samples that had no diagnosis of PPH or PE. PDGF-BB protein levels were assessed by ELISA and Western blot methods. Logistic regression analysis was used to evaluate the association between PDGF-BB and PPH. Please refer to supplemental materials for detailed methods (below). Table 1 below7shows the demographic characteristics of the study subjects.

[0127] Table 1. Demographic characteristics of study subjects.

[0128] Control PPH PE +PPH Characteristic PE (n=41) P value (n=10) (n=23) (n=6) Maternal age at delivery

[0129] 29.3±5.4 29.5+5.0 31.1+5.6 32.8±6.9 0.44 (years)

[0130] Maternal race 0.043

[0131] Asian 0 0 1 (2.4) 0

[0132] Black or African American 1 (10) 2 (8.7) 4 (9.4) 4 (66.7)

[0133]

[0134] Docket No. 650053.01262

[0135] Control PPH PE +PPH Characteristic PE (n=41) P value (n=10) (n=23) (n=6) White 9 (90) 21 (91.3) 35 (85.4) 2 (33.3) American Indian or Alaska

[0136] 0 0 1 (2.4) 0

[0137] Native

[0138] Hispanic or Latino 0 0 1 (2.4) 0 0.43 Gestational age of sample 27.3 (26.3, 26.7 (25.7, 27.7 (25.9, 29.4 (26.3,

[0139] 0.42 (week) 28.1) 28.4) 31.0) 36.0)

[0140] 24.4 (21.4, 36.9 (29.9, 30.4 (23 9, 36.9 (29.9, BMI (pre-pregnancy) 0.047

[0141] 27.8) 43.2) 36.3) 43.2) Gestational diabetes 0 3 (13) 5 (12.2) 1 (16.7) 0.69 Gestational hypertension 0 4 (17.4) 13 (31.7) 1 (16.7) 0.14 Anemia 0 0 0 1 (16.7) 0075 Placental previa 0 2 (8.7) 0 0 0.31 Polyhydraminos 0 1 (4.4) 1 (2.4) 0 >0.99 Placental abrupti on 0 1 (4.4) 0 0 0.49

[0142]

[0143] Macrosomia 0 0 0 1 (16.7) 0.075

[0144] Results

[0145] Among the 80 plasma samples, body mass index was statistically significantly different among PPH, PPH+PE, PE and controls. >i' 047 (Table 1). ELISA assay results showed that PDGF-BB levels were significantly lower in PPH (median and interquartile range [7.5 (6.6, 9.3) pg / mL] and PPH+PE [6.7 (4.8, 10.8) pg / mL] compared to controls [22.9 (21.7, 24.5) pg / mL], adjusted ><0.0001 and p=0.0015, respectively. PE alone group (22.3 pg / mL) showed no significant difference from controls (FIG. 3A). Western blot method confirmed ~l-fold reduction in PDGF-BB in PPH groups (FIG 3B). Higher PDGF-BB levels were inversely associated with PPH risk [OR 0.62, 95% CI: 0.44-0.88; >=0.0068]. ROC analysis identified a PDGF-BB cutoff of 11.5 pg / mL with 100% sensitivity7and specificity (AUC=1.0) (FIG. 3C). ELISA results are shown in Table 2.

[0146] Table 2: Plasma PDGF-BB levels in pregnant subjects.

[0147] Gestation Age BMI (pre at Collection PDGF-BB Age Ethnicity7Race pregnancy) Diagnosis Comments (Weeks, Davs) (pg / mL)

[0148]

[0149] PPH 34 NH W 27.73 CBD; GD 27, 4 8 23Docket No. 650053.01262

[0150] Gestation Age BMI (pre at Collection PDGF-BB Age Ethnicity Race pregnancy) Diagnosis Comments (Weeks, Days) (pg / mL) 27 NH W 48.74 CBD 28.3 5.84 28 NH W 22. J 1 CBD 27, I 5 53 30 NH w 19.92 CBD; PP 26, 5 6.62 NH B 19.53 CBD; PP 21, 6 6.40 20 NH W 23.99 CBD 27, 1 6.59

[0151] PPH,

[0152] APGAR <7

[0153] 5.34 at 5 mm upon

[0154] 33 NH w 39.32 CBD; GD delivery 27. 5

[0155] 29 NH w 31.89 CBD; PH 25, 5 5.75 27 NH w 30.38 CBD 25, 4 7.48

[0156] Placenta

[0157] abruption and

[0158] 11.31 PPH upon

[0159] 33 NH w 22.63 CBD delivers' 26. 1

[0160] 24 NH w 39.31 CBD; GH 24, 6 9.21 37 NH B 29.78 CBD 35, 1 8.10 35 NH W 25.25 CBD; GH 37, 6 7.18 29 NH w 25.79 CBD 27.4 944 29 NH w 22.11 CBD 26. 0 7.38 27 NH w 20.66 CBD 29 2 7.28 33 NH w 38.35 CBD; GH 24, 4 8.23 30 NH w 22.86 CBD 25, 5 7.31 23 NH w 26.09 CBD 28, 5 9 32 39 NH w 40.25 CBD; GH 23, 2 9.31 NH w CBD; GD 28, 5 10.57 NH w CBD 26, 3 7.80 NH w CBD 26, 0 9.26 39 NH w 35.27 CBD 38, 2

[0161] 20 NH w 34.38 CBD 25, 5 21.97 27 NH w 23.4 CBD; GH 29, 0 26.96 41 II w 38.1 CBD 33.2 18.14 29 NH w 34.38 CBD 24, 2 23 86 PE 28 NH w 24.17 CBD 26, 1 21.60

[0162] CBD;

[0163] Diabetes

[0164] meliitus

[0165] complicating

[0166] 33 NH w 37.2 pregnancy 26, 6 22.79

[0167]

[0168] 34 NH w 28.66 CBD; GH 25, 0 26.55Docket No. 650053.01262

[0169] Gestation Age BMI (pre at Collection PDGF-BB Age Ethnicity Race pregnancy) Diagnosis Comments (Weeks, Days) (pg / mL) 28 NH W 34.7 CBD 28.6 23 05 29 NH W 18.65 CBD 30, 4 2291 34 NH w 30.21 CBD; GH 36, 5 23.80

[0170] CBD; GD;

[0171] 32 NH w 25.68 GH 25, 6

[0172] 29 NH A 37.73 CBD; GH 26, 0 21.51 30 NH w 23.08 CBD 27, 6 20.55 27 NH w 27.11 CBD; GH 24.3 2222 33 NH w 35.84 CBD; GH 33. 3 2475 30 NH w 32.06 CBD 26, 3 19.51 30 NH w 34.38 CBD 27, 5 22.28 32 NH w 36.27 CBD 36, 2 20.66 36 NH w 39.48 CBD 36. 3 2024 39 NH w 39.7 CBD; GH 26. 2 23 1 i 34 NH w 21.96 CBD; GH 28, 2 24.14

[0173] CBD; GD;

[0174] 30 NH w 38.73 GH; PH 31, 2 24.98 36 NH w 26.96 CBD; GH 36, 3 26.58 27 NH B 27.27 CBD; IUGR 26, 5 20.38 35 NH w 31.12 CBD 28.0 22 17 37 NH B 43.77 CBD; GH 25, 5 23 91 35 NH w 23.86 CBD 27, 2 23.77 45 NH B 42.22 CBD 24, 5 17.31 32 NH B 42.91 CBD 25, 1 19.51 18 NH W 20.17 CBD 31.0 2001 22 NH w 43.93 CBD; GD 25, 4 23 51 29 NH w 22.89 CBD 25, 2 13.90 30 NH w 23.23 CBD 27, 2 23.57 33 NH w 31.64 CBD 26, 3 22.28 24 NH w 19.22 CBD 27. 5 21 85 35 NH w 19.3 CBD 24, 1 25 73 25 NH w 22.96 CBD 39, 1 26.78 33 NH Al 30.38 CBD; GD 38, 0 16.95 21 NH w 30.12 CBD; GD 28, 5 19.56 35 NH w 28.97 CBD; GH 29, 6 12.41

[0175]

[0176] 29 NH B 27.8 CBD 30, 6 12.94Docket No. 650053.01262

[0177] Gestation Age BMI (pre at Collection PDGF-BB Age Ethnicity Race pregnancy) Diagnosis Comments (Weeks, Days) (pg / mL)

[0178] Macrosomia,

[0179] PPH upon

[0180] delivery,

[0181] postpartum

[0182] PE diagnosed

[0183] 43 NH W 43.16 CBD; GA PPD#8 21, 3 6 69

[0184] CBD; GD;

[0185] PE + 33 NH B 29.91 GH 36, 2 4.61 PPH 39 NH B 42.11 CBD 36.0 10.78 25 NH W 31.62 CBD; HC 28. 0 4.78

[0186] Preterm (< 37

[0187] weeks), PPH,

[0188] & PE w /

[0189] severe

[0190] features

[0191] 28 NH B 57.4 CBD upon delivery' 26. 2 6.62 29 NH W 34.92 CBD 28, 3 24.46 28 NH W 27.79 CBD 11.72 25 NH w 26.31 CBD 26, 2 2544 21 B 24.97 CBD 24, 4 2299 32 NH W 19.91 CBD 26. 0 1839 Control

[0192] 36 NH w 23.33 CBD 26, 0 21.71 97 NH w 23.86 CBD 28, 4 22.88 31 H w 40.74 CBD 28, 0 22.08 39 NH w 21.43 CBD 27, 3 25 73

[0193]

[0194] 25 NH w 18.47 CBD 28, 1 23.48 *Noles: PPH = Postpartum Hemorrhage; PE = Preeclampsia, NH = Non-Hispanic; H = Hispanic or Latino; W = White; B = Black or African American; Al = American Indian or Alaska Native; A = Asian; CBD = Cord Blood Donor; GD = Gestational Diabetes for specified pregnancy; PP = Placenta Previa for specified pregnancy; PH = Polyhydramnios; GH = Gestational Hypertension for specified pregnancy; IUGR = Intrauterine Growth Restriction; GA::::Gestational Anemia; HC:::Hepatitis C

[0195] Statistical analysis was also performed to test the interaction between PDGF-BB and trimester. While sample sizes were small (2ndtrimester Control+PPH n=22; 3rdtrimester Control+PPH n:::10), the analysis did not demonstrate a significant difference (P:::0.95; Tables.3 and 4). Samples collected between weeks (>14 and <28) were grouped as 2ndtrimester and weeks >28 were grouped as 3rdtrimester. The results were similar for both trimesters.Docket No. 650053,01262

[0196] Table 3. Gestational age (by trimester) for the samples analyzed.

[0197] Trimester Frequency Percent Cumulative Cumulative Percent Frequency

[0198] 2nd 47 59.49 47 59.49

[0199] 3rd 32 40.51 79 100

[0200] Frequency Missing = 1

[0201] Trimester by Group (All)

[0202] Trimester Group

[0203] Control PE PE+PPH PPH Total 2nd 5 23 2 17 47

[0204] 10.64 48.94 4.26 36.17

[0205] 55.56 56.10 33.33 73.91

[0206] 3rd 4 18 4 6 32

[0207] 12.5 56.25 12.5 18.75

[0208] 44.44 43.9 66.67 26.09

[0209] Total 9 41 6 23 79 Frequency Missing = 1

[0210] Trimester by Group (Control v. PPH)

[0211] Trimester Group

[0212] Control PPH Total 2nd 5 17 22

[0213] 22.73 77.27

[0214] 55.56 73.91

[0215] 3rd 4 6 10

[0216] 40 60

[0217] 44.44 26.09

[0218] Total 9 23 32

[0219]

[0220] Frequency Missing = 1

[0221] *Notes: in cells with three values, reported from top to bottom are: frequency, row percentage, and column percentage.

[0222] Table 4. Logistic regression (PPH vs Control) testing the interaction of PDGF-BB levels and trimester.

[0223] Response Profile

[0224] Ordered Value Group Total Frequency

[0225] 1 Control 9

[0226] 2 PPH 23

[0227] Type 3 Analysis of Effects

[0228] Effect DF Wald Pr >

[0229] Chi- ChiSq

[0230] Square

[0231] PDGF-BB levels 1 9.1431 0.0025

[0232]

[0233] Trimester 1 0.0019 0.9655Docket No. 650053,01262

[0234] PDGF-BB 1 0.0033 0.9541

[0235] levels*trimester

[0236]

[0237] *Notes: probability modeled is Group = ‘" PPI C

[0238] For the 2ndtrimester (samples collected between weeks >14 and <28), for every unit increase in PDGF-BB, the likelihood of developing PPH was decreased by 31.5%, odds ratio (OR) and 95% confidence interval (CI) 0.685, 0.510-0.921. P=0.012 (Table 5; FIG. 4).

[0239] Table 5. Logistic regression of samples collected during the 2ndtrimester of pregnancy (PPH vs. control).

[0240] Response Profile

[0241] Ordered Value Group Total Frequency

[0242] 1 Control 5

[0243] 2 PPH 17

[0244] Analysis of Penalized Maximum Likelihood Estimates

[0245] Parameter DF Estimate Standard Wald Chi- Pr > ChiSq Error Square

[0246] Intercept 1 6.0125 2.1118 8.1064 0.0044 PDGF- 1 0.1505 62984 0.0121 BB levels 0.3777

[0247] Odds Ratio Estimates

[0248] Effect Point Estimate 95% Wald Confidence

[0249] Limits

[0250]

[0251] PDGF-BB levels 0.685 0.51 0.921 *Notes: probability modeled is Group = " PPH”

[0252] For the 3rdtrimester (samples collected during weeks >28), for ever)- unit increase in PDGF-BB, the likelihood of developing PPH was decreased by 23.2%, odds ratio (OR) and 95% confidence interval (Cl) 0.768, 0.590-0.999, P=0.049 (Table 6; FIG. 5).

[0253] Table 6. Logistic regression of samples collected during the 3rdtrimester of pregnancy (PPH vs, control).

[0254] Response Profile

[0255] Ordered Value Group Total Frequency

[0256] 1 Control 4

[0257] 2 PPH 6

[0258] Analysis of Penalized Maximum Likelihood Estimates

[0259] Parameter DF Estimate Standard Wald Chi- Pr > ChiSq Error Square

[0260] Intercept 1 4.0709 2.1075 3.7311 0.0534 PDGF-BB 1 -0.2645 0.1344 3.8707 0.0491

[0261]

[0262] levelsDocket No. 650053,01262

[0263] Odds Ratio Estimates

[0264] Effect Point Estimate 95% Wald Confidence

[0265] Limits

[0266]

[0267] PDGF-BB levels 0.768 0.59 0.999 *Notes: probability modeled is Group ‘ PPIT’

[0268] Discussion

[0269] The results demonstrate a significant 3-fold decrease in PDGF-BB levels m the plasma of patients diagnosed with PPH when compared to control patients. PDGF-BB has been studied as a biomarker in other medical conditions such as postpartum depression (PPD)nand obesity / metabolic syndrom13that could share underlying pathophysiological mechanisms with PPH. Limitations of this study include the retrospective nature of the analysis on limited sample sets, selection bias to the analysis due to samples from a single pregnancy center and the lack of clarity of when PDGF-BB level drop in pregnancy is predictive of PPH.

[0270] Conclusions

[0271] Low antepartum plasma PDGF-BB levels were significantly associated with PPH and demonstrated high diagnostic performance at a defined threshold. These findings support PDGF-BB as a promising early biomarker for PPH risk stratification. Prospective validation in larger, diverse cohorts is warranted to assess clinical applicability and potential integration into management of obstetric hemorrhage.

[0272] Materials and Methods

[0273] Ethical considerations-. This study was approved by the Institutional Review Board (IRB) of the Medical College of Wisconsin titled, "‘Cilia protein expression changes in patients with normal delivery and those diagnosed with preeclampsia”.

[0274] Study Design and Participants’. This study was conducted to investigate the potential of PDGF-BB as a diagnostic marker for PPH. Blood samples were collected from pregnant women antepartum, between 21 to 40 w eeks of gestation, at a single medical center. A total of 80 samples were included in the study, with participants categonzed into four groups based on their pregnancy status and clinical diagnoses. The groups consisted of 23 samples from patients who later developed PPH, 6 samples from patients with both PPH and preeclampsia (PE), 41 samples from patients diagnosed with PE, and 10 samples from healthy control patients with uncomplicated pregnancies.

[0275] Inclusion and Exclusion Criteria'. A retrospective study was conducted using biospecimens from the Medical College of Wisconsin’s Maternal Placenta & Cord (MPC) Bank. Participants in the MPC Bank are pregnant individuals aged >18 years who can read andDocket No. 650053.01262

[0276] speak English and provide informed consent. Enrollment includes a one-time maternal blood collection during routine care and permission to store any residual blood or placental tissues otherwise discarded after delivery, resulting in a large, heterogeneous biobank.

[0277] For this study, a convenience sample of 80 participants (0.6% of total banked samples) was selected. Eligible cases included pregnant individuals with a maternal blood sample collected at 27 ± 3 weeks gestation and a diagnosis at delivery of: postpartum hemorrhage (PPH, n:::23), preeclampsia (PE, n:::41). both PE and PPH (n:::6). or neither (controls. n:::10). Case samples (PE, PPH, and PE+PPH) were collected between 2016 and 2021; control samples were collected in 2022. The MPC Bank housed 10,738 samples by 2021 and expanded to 12,680 samples by 2022.

[0278] Sample Collection and Processing: Blood plasma samples were received from the MCW tissue bank. Briefly, blood samples were collected via venipuncture into CTAD (citrate-theophylline-adenosine-dipyridamole) tubes with optional prostaglandin El or apyrase (platelet-activation inhibitors) to suppress platelet activation. Samples were processed within 30 minutes of collection through a two-step centrifugation protocol (low-g spin followed by >10,000 g) to yield platelet-free plasma (PFP). This rapid, time-stamped workflow allows for the measurement of circulating PDGF-BB, rather than artifactual platelet-released PDGF-BB. Processed samples were stored at -80°C until analysis. All sample collection procedures were conducted m accordance with institutional guidelines and ethical standards.

[0279] Western Blot Analysis: To qualitatively assess PDGF-BB protein expression. Western blot analysis was performed. Plasma samples (20 pL) were subjected to protein quantification using the BioRad DC protein assay followed by detection in a SpectraMax 340PC absorbance microplate reader. The plasma samples were diluted with RIPA buffer (Sigma) with complete mini EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland) and PhosSTOP phosphatase inhibitor (Roche) using a Qiagen TissueRuptor (Hilden, Germany). 20 pg of protein was loaded in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). Membranes were incubated overnight at 4°C with a primary antibody specific to PDGF-BB (1:500, Novas Biologicals, Cat#NBP 1-58279), and p-actin (1: 1000, Cell signaling technology, Cat# 8457) followed by incubation with anti-rabbit horseradish peroxi dase-conjugated secondary antibody (1:1000, Cell signaling technology, Cat# 7074). The bands were visualized using enhanced chemiluminescence (ECL) detection, and band intensity was quantified using ImageJ software.Docket No. 650053.01262

[0280] Enzyme-Linked Immunosorbent Assay (ELISA)-. Quantitative analysis of PDGF-BB protein levels was performed using a commercially available ELISA kit (RayBiotech, Norcross, GA, Cat# ELH-PDGFBB-1 ). Plasma samples were thawed and diluted according to the manufacturer’s instructions. A standard curve was generated using known concentrations of recombinant PDGF-BB, and sample concentrations were calculated by comparing their absorbance values at 450 nm to the standard curve according to an Excel file template provided by the company. All samples were measured in duplicate to ensure accuracy, and the interassay coefficient of variation (CV) was <10%.

[0281] Statistical Analysis: Descriptive statistics were used to summarize the baseline characteristics of the study groups. All data are presented as n (%) or mean ± standard deviation (SD) or median and interquartile range. Shapiro-Wilk test was used to test normality and Levene’s test was used to check the homogeneity of variance assumption. Demographics were compared among the groups by analysis of variance (ANOVA), Kruskal-Wallis test or Fisher’s exact as appropriate. For group comparisons of PDGF-BB levels from western blot or ELISA, a one-way ANOVA or Kruskal-Wallis test was used followed by Dunnett test or Dunn’s test to adjust for multiple comparisons. Logistic regression was performed to examine the association between PDGF-BB levels and development of PPH in PPH patients and controls. Firth’s penalized likelihood approach was used to address issues of separability. A p-value of <0.05 was considered statistically significant. Statistical analysis was performed using SAS version 9.4 (SAS Institute Inc.. Cary, NC) or SPSS version 29.0 (IBM Corp., Armonk, NY).

[0282] REFERENCES

[0283] 1. Say, L., D. Chou, A. Gemmill, Ö Tunçalp, A. B. Moller, J. Daniels, A. M. Gülmezoglu, M. Temmerman, and L. Alkema. 2014. " Global causes of maternal death: a WHO systematic analysis." Lancet Glob Health 2 (6):e323-33.

[0284] 2. Main EK, Goffman D, Scavone BM, et al. National Partnership for Maternal Safety: Consensus Bundle on Obstetric Hemorrhage [published correction appears in Obstet Gynecol.

[0285] 2015 Nov;126(5):llll] [published correction appears in Obstet Gynecol. 2019 Jun;133(6):1288.

[0286] 3. Callaghan, W. M., E. V. Kuklina. and C. J. Berg. 2010. " Trends in postpartum hemorrhage: United States, 1994-2006." Am J Obstet Gynecol 202 (4):353.el-6.

[0287] 4. Corbetta-Rastelli, C. M., A. M. Friedman, N. C. Sobhani. B. Arditi, D. Goffman, and T. Wen. 2023. " Postpartum Hemorrhage Trends and Outcomes in the United States, 2000-2019." Obstet Gynecol 141 (1): 152-161.Docket No. 650053.01262

[0288] 5. Committee on Practice Bulletins-Obstetrics. Practice Bulletin No. 183: Postpartum Hemorrhage. Obstet Gynecol. 2017 Oct;130(4):el68-el86. doi: 10.1097 / AOG.0000000000002351. PMID: 28937571.

[0289] 6 Dilla, A. J., J. H Waters, and M. H Yazer. 2013. ‘’Clinical validation of risk stratification criteria for peripartum hemorrhage.’’ Obstet Gynecol 122 (1): 120-126.

[0290] 7. Kramer, M. S., C. Berg, H. Abenhaim, M. Dahhou, J. Rouleau, A. Mehrabadi, and K. S. Joseph. 2013. " Incidence, risk factors, and temporal trends in severe postpartum hemorrhage." Am J Obstet Gynecol 209 (5):449.el-7.

[0291] 8. Gupta A, Thirugnanam K, Thamilarasan M, et al. Cilia proteins are biomarkers of altered flow in the vasculature. JCI Insight. 2022;7(6):el51813. Published 2022 Mar 22. 9 Thirugnanam K, Prabhudesai S, Van Why E, et al. Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability. Biochem Pharmacol. 2022:202:115143.

[0292] 10. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999;79(4): 1283-1316.

[0293] 11. Idemoto K, Ishima T, Niitsu T, et al. Platelet-derived growth factor BB: A potential diagnostic blood biomarker for differentiating bipolar disorder from major depressive disorder. J Psychiatr Res. 2021; 134:48-56.

[0294] 12. Zhou J, Deng Y, Yan L, Zhao H, Wang G; China HepB-Related Fibrosis Assessment Research Group. Serum platelet-derived growth factor BB levels: a potential biomarker for the assessment of liver fibrosis in patients with chronic hepatitis B. Int J Infect Dis. 2016:49:94- 99.

[0295] 13. Tisato V, Toffoli B, Monasta L, et al. Patients affected by metabolic syndrome show- decreased levels of circulating platelet derived growth factor (PDGF)-BB. Clin Nutr.

[0296] 2013;32(2):259-264.

[0297] 14. Cheng N, Liu Y, Mukama O, et al. A signal -enhanced and sensitive lateral flow aptasensor for the rapid detection of PDGF-BB. RSC Adv. 2020; 10(32): 18601-18607. Published 2020 May 18.

[0298] 15. Evensen et al, American Family Physician, 2017: (95)7: 442-449.

[0299] 16. Bienstock et al.. Postpartum Hemorrhage. N Engl J Med 2021:384: 1635-45.

[0300] 17. Gaengel et al., Endothelial-mural cell signaling in vascular development and angiogenesis. Arterioscler Thromb Vase Biol 2009;29:630-8.

Claims

1. Docket No. 650053.01262CLAIMSWhat is claimed:

1. A biomarker for use in diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) in a pregnant subject wherein the biomarker is Platelet-Derived Growth Faclor-BB (PDGF-BB), and wherein the PDGF-BB is present in the subject's serum or plasma at or below a threshold concentration or a control concentration, optionally wherein the threshold concentration is about 11.5 pg / mL or 0.5 pg / mL.

2. A method of sample processing, comprising:(a) producing a fraction of a biological sample obtained from a subject, comprising:(i) introducing the biological sample to an antibody under conditions sufficient to bind the antibody to an analyte in the biological sample, wherein the antibody is immobilized to a solid support, and(ii) selectively removing components of the biological sample that are not bound to the antibody; and(b) quantifying the analyte within the fraction.wherein the biological sample is a blood sample, a serum sample, or a plasma sample, wherein the subject is pregnant, and wherein the analyte is Platelet-Derived Growth Factor-BB (PDGF-BB).

3. The method of claim 2, comprising performing (a)-(b) using an enzyme-linked immunoassay (ELISA).

4. The method of claim 3. wherein the ELISA is a single-molecule ELISA.

5. The method of any one of claims 2-4, wherein a quantity of the analyte resulting from the quantifying in (b) indicates a presence of or a risk of developing postpartum hemorrhage, when the quantity is lower than a threshold concentration or a control concentration.6 The method of any one of claims 2-5, wherein the biological sample is obtained during the first trimester of pregnancy, during the second trimester of pregnancy, during the third trimester of pregnancy, during labor for childbirth, and / or immediately following childbirth.Docket No. 650053.012627. The method of claim 5 or 6, wherein the threshold concentration is about 0.5 pg / mL, optionally wherein the biological sample is obtained during labor for childbirth or immediately following childbirth.

8. The method of claim 5 or 6, wherein the threshold concentration is about 11.5 pg / mL, optionally wherein the biological sample is obtained during the second or third trimester of pregnancy.

9. The method of any one of claims 2-8, wherein the method has a lower limit of quantification (LLOQ) for PDGF-BB, and wherein the LLOQ is less than about 0.5 pg / mL.

10. A method of treatment, comprising:(a) quantifying the biomarker of claim 1 in a biological sample obtained from the pregnant subject;(b) administering a treatment for postpartum hemorrhage, when a quantity of the biomarker is lower than the threshold concentration or the control concentration.

11. The method of claim 10, wherein the biological sample is obtained during the first trimester of pregnancy, during the second trimester of pregnancy, during the third trimester of pregnancy, during labor for childbirth, and / or immediately following childbirth.

12. The method of claim 10 or 11, wherein the threshold concentration is about 0.5 pg / mL, optionally wherein the biological sample is obtained during labor for childbirth or immediately following childbirth13. The method of claim 10 or 11, wherein the threshold concentration is about 11.5 pg / mL, optionally wherein the biological sample is obtained during the second or third trimester of pregnancy.

14. The method of any one of claims 10-13, wherein the quantify ing is performed using an enzyme-linked immunoassay (ELISA).

15. The method of claim 14, wherein the ELISA is a single-molecule ELISA.

16. The method of any one of claims 10-15, wherein the biological sample is a blood sample, a serum sample, or a plasma sample.Docket No. 650053.0126217. The method of any one of claims 10-16, wherein the treatment for postpartum hemorrhage comprises administering a uterotonic agent, administering a blood product, performing a tamponade procedure, performing controlled cord contraction and / or active management of the second stage of labor, performing a surgical procedure, and / or performing a uterine massage, optionally wherein the surgical procedure comprises dilation and curettage, interventional radiology guided embolization, and / or peripartum hysterectomy.

18. The method of claim 17, wherein the uterotonic agent is selected from the group consisting of oxytocin, methylergonovine, 15-methyl prostaglandin F2a, misoprostol, and combinations thereof.

19. A kit for detecting Platelet-Derived Growth Factor-BB (PDGF-BB) in a biological sample obtained from a pregnant subject, the kit comprising:(a) a PDGF-BB-specific antibody, wherein the antibody is immobilized to a bead or ELISA plate: and(b) instructions for performing an ELISA assay.

20. The kit of claim 19, wherein the biological sample is a blood sample, a serum sample, or a plasma sample.

21. The kit of claim 19 or 20, further comprising instructions for calculating a relative risk that the subject will develop postpartum hemorrhage.

22. A method for diagnosing or predicting a risk of developing postpartum hemorrhage (PPH) comprising:(a) obtaining a plasma or serum sample from a pregnant subj ect at any time during pregnancy or following childbirth;(b) detecting Platelet-Derived Growth Factor-BB (PDGF-BB) in the sample; and (c) determining a concentration of the PDGF-BB in the sample, wherein the concentration is about 11.5 pg / mL or less.

23. The method of claim 22, wherein the sample is obtained during the first trimester of pregnancy, during the second trimester of pregnancy, during the third trimester of pregnancy, during labor for childbirth, and / or immediately following childbirthDocket No. 650053.0126224. The method of claim 22 or 23, wherein the concentration is about 0.5 pg / mL or less, optionally wherein the sample is obtained during labor for childbirth or immediately following childbirth..

25. The method of any one of claims 22-24, wherein the concentration of PDGF-BB is within a range of about 1 fg / mL to about 11.5 pg / mL.

26. The method of any one of claims 22-25, wherein the quantifying is performed using an enzyme-linked immunoassay (ELISA).

27. The method of claim 26, wherein the ELISA is a single-molecule ELISA.

28. The method of any one of claims 2-18 or 22-27, further comprising detecting and / or quantifying one or more marker of cilium.

29. The method of claim 28, wherein the one or more marker of cilium comprises ARL 13B, y-tubulin, and / or IFT88.

30. The method of any one of claims 2-18 or 22-29, wherein the biological sample is a platelet-free plasma sample.

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