Circulating biomarkers for placental or fetal health

文档序号：863412 发布日期：2021-03-16 浏览：5次中文

阅读说明：本技术 用于胎盘或胎儿健康的循环生物标志物 (Circulating biomarkers for placental or fetal health ) 是由斯蒂芬·唐图海瓦哈·乔伊·利诺特蕾莎·玛丽·麦克唐纳苏珊·菲利帕·沃克于 2019-05-24 设计创作，主要内容包括：公开了有助于临床妊娠管理的测定、方案和试剂,其改善了在胎盘功能不全环境中发育的胎儿的产前和产后健康结果。(Assays, protocols, and reagents are disclosed that facilitate clinical pregnancy management that improve prenatal and postpartum health outcomes for a fetus developing in a placental insufficiency environment.)

1. An assay to determine placental health status in a female mammalian subject, the method comprising determining circulating levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in the ratio relative to a control or a change in time is indicative of placental insufficiency, and an increase in SPINT-1 and/or SYNDECAN-1 is a measure of adequate placental function or a measure of improvement in adequate placental function.

2. An assay to detect abnormal fetal weight, such as a giant child, in a female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein an increase in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or an increase over time or a change in the ratio relative to a control or a change over time is indicative of a potential giant child.

3. An assay to detect abnormal fetal weight in a female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein an increase in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or an increase in the ratio relative to a control or a change in time is indicative of potentially high fetal weight, and a decrease in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in the ratio relative to a control or a change in time is indicative of potentially low fetal weight or less gestational age or fetal growth restriction.

4. The assay according to any one of claims 1 to 3 comprising determining the level of SPINT 1.

5. The assay of any one of claims 1-3, comprising determining a level of SYNDECAN-1.

6. The assay of any one of claims 1 to 3, comprising determining the level of SPINT1 and SYNDECAN-1.

7. The assay according to any one of claims 1 to 6, which comprises determining the level of SPINT1 and/or SYNDECAN-1 and at least one other biomarker and/or physiochemical parameter and/or clinical risk factor.

8. The assay of claim 7, wherein the at least one other biomarker is one or more of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt), and/or Vascular Endothelial Growth Factor (VEGF).

9. The assay of claim 7, wherein the physiochemical parameter is generated by ultrasound or physical measurement.

10. The assay according to any one of claims 1 to 9, wherein the level or ratio of levels of SPINT1 and/or SYNDECAN-1 is analyzed by an algorithm or by an analytical function or analytical method or other data processing means.

11. The assay of any one of claims 1-10, further comprising assessing the subject for one or more risk factors associated with increased risk of abnormal fetal weight.

12. The assay according to any one of claims 1 to 11, wherein the mammalian subject is a human female.

13. The assay according to any one of claims 1 to 12, wherein circulating SPINT1 and/or SYNDECAN-1 in maternal whole blood, plasma or serum is determined.

14. The assay of any one of claims 1 to 13, wherein the pregnancy of a subject determined to have placental insufficiency, or a subject in which a large fetus is predicted, is clinically managed to maximize the health outcome of the fetus.

15. The assay of claim 14, wherein the management comprises preterm delivery of a fetus.

16. A clinical management regimen for a pregnant mammalian subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein a decrease in the level of SPINT1 and/or SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 and/or SYNDECAN-1 is indicative of placental insufficiency, and wherein the fetus is monitored or has undergone preterm delivery.

17. A clinical management protocol for a neonate in a pregnant human subject, the protocol comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein an increase in the SPINT1 and/or SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting an increase in the level of SPINT1 and/or SYNDECAN-1 is indicative of a neonate, wherein the fetus is monitored and is likely to undergo early delivery.

18. The protocol of claim 16 or 17, comprising determining a level of SPINT 1.

19. A scheme according to claim 16 or 17, comprising determining a level of SYNDECAN-1.

20. A scheme according to claim 16 or 17, comprising determining the levels of SPINT1 and SYNDECAN-1.

21. A regimen according to any one of claims 16 to 20, which comprises determining the level of SPINT1 and/or SYNDECAN-1 and at least one other biomarker and/or physiochemical parameter.

22. The regimen according to claim 21, wherein at least one other biomarker is one or more of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt), and/or Vascular Endothelial Growth Factor (VEGF).

23. The protocol of claim 21, wherein the physiochemical parameter is generated by ultrasound or physical measurement.

24. The protocol of any one of claims 16 to 23, wherein the level or ratio of levels of SPINT1 and/or SYNDECAN-1 is analyzed by an algorithm or by an analytical function or analytical method or other data processing means.

25. The regimen according to any one of claims 16 to 24, wherein the mammalian subject is a human female.

26. The regimen according to any one of claims 16 to 25, wherein circulating SPINT1 and/or SYNDECAN-1 in maternal whole blood, plasma or serum is determined.

27. The regimen according to any one of claims 16 to 26, which is in the management of a Fetal Growth Restriction (FGR) or Small for Gestational Age (SGA) infant or giant fetus.

28. An assay for determining the placental functional sufficiency state in a subject, the assay comprising determining the concentration of a biomarker selected from the group consisting of SPINT1 and SYNDECAN-1 in a circulating biological sample from the subject; subjecting said levels to an algorithm or analytical function or analytical method or other data processing means generated from a first knowledge base of data comprising levels of the same biomarkers from subjects or cohorts of subjects having a known status with respect to sufficiency of placental function, wherein said algorithm or analysis or data processing provides an index of probability that a subject has or does not have placental insufficiency or placental sufficiency.

29. Use of a training data knowledge base to generate an algorithm or analytical function or analytical method or other data processing means, said training data knowledge base comprising biomarker levels selected from the group consisting of: either SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1 with at least one other biomarker, said algorithm or analytical function or analytical method or other data processing means providing a probability index predicting a property of the placenta after inputting a second knowledge base of data comprising levels of the same biomarker from a patient with unknown placental functional sufficiency state.

30. A panel of biomarkers for detecting placental insufficiency or placental health state or dysfunction in a subject, the panel comprising agents that specifically bind to a biomarker to determine the level of said biomarker and then optionally subjecting said level to an algorithm or analytical function or analytical method or other data processing means generated from a first data repository, said biomarker selected from both SPINT1, SYNDECAN-1, SPINT1 and SYNDECAN-1 or one of SPINT1 or decan-1 with at least one other biomarker, said first data repository comprising levels of the same biomarker from subjects having a known state with respect to a condition, wherein said algorithm or analysis or data processing provides a probability index that a subject has or does not have placental insufficiency.

31. A method for monitoring pregnancy progression in a patient, the method comprising:

(a) providing a sample of circulating fluid from the patient;

(b) determining the level of SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1 with at least one other biomarker and subjecting said level to an algorithmic or analytical function or analytical method or other data processing means to provide an index of probability that the patient has adequate or inadequate placental function; and

(c) repeating steps (a) and (b) at a later point in time and comparing the result of step (b) with the result of step (c), wherein a difference in the probability index indicates the progression of placental health.

32. The method of claim 28 or the use of claim 29 or the group of claim 30 or the method of claim 31, further comprising inputting physiochemical data generated by ultrasound or physical measurements.

33. The method, use or group of claim 32, further comprising inputting one or more risk factors associated with increased risk of abnormal fetal weight.

34. A method of detecting placental health status in a subject, the method comprising:

(a) providing a sample of circulating fluid from a patient;

(b) extracting nucleic acid molecules from the sample or cells within the sample, the nucleic acid molecules comprising mRNA encoding a biomarker or a portion thereof;

(d) determining the level of mRNA encoding the biomarker; and

(e) subjecting the levels of the biomarkers to an algorithm or analytical function or analytical method or other data processing means which provides an index of the probability that the patient has adequate or insufficient placental function.

35. A method of allowing a user to determine a status of a subject regarding a level of placental sufficiency or placental insufficiency, the method comprising:

(a) receiving data from the user via a communication network in the form of levels or concentrations of SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1, or one of SPINT1 or SYNDECAN-1 with at least one other biomarker;

(b) processing the subject's data via an algorithm or analytical function or analytical method or other data processing means, which provides a disease index value;

(c) determining a status of the subject according to a result of the comparison of the disease index value to a predetermined value; and

(d) communicating an indication of the status of the subject to the user via the communication network.

36. A diagnostic device which, when used in the assay or protocol of any one of claims 1 to 27, monitors the level of placental functional sufficiency.

37. An assay for determining the status of a giant fetus in a subject, the assay comprising determining the concentration of a biomarker selected from the group consisting of SPINT1 and SYNDECAN-1 in a circulating biological sample from the subject; subjecting the levels to an algorithm or analytical function or analytical method or other data processing means generated from a first data repository comprising levels of the same biomarker from a subject or cohort of subjects having a known status with respect to a large fetus, wherein the algorithm or analysis or data processing provides an index of probability of an infant of the subject with or without a large fetus.

38. A composition comprising a sample obtained from a pregnant mammalian subject and an antibody that specifically binds to SPINT1 or SYNDECAN-1.

39. The composition of claim 38, comprising an antibody that specifically binds to SPINT1 and an antibody that specifically binds to SYNDECAN-1.

40. The composition of claim 38 or claim 39, wherein the sample is a circulating maternal fluid.

41. The composition of claim 40, wherein the circulating maternal fluid comprises whole blood, plasma, or serum.

FIELD

The present invention relates generally to protocols for clinically managing pregnancy in mammals, including humans. The present invention provides diagnostic targets and reagents that facilitate clinical pregnancy management, thereby enabling improved prenatal (pre-natal) and postnatal (post-natal) health outcomes for fetuses developing in a placental insufficiency (placental insufficiency) environment.

Background

Bibliographic details of the publications cited by authors in this specification are collected alphabetically in the final part of the specification.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Fetal Growth Restriction (FGR), also known as intrauterine growth restriction (IUGR), represents a major cause of poor postpartum health, is a major cause of stillbirth in humans, and leaves a life-long problem for children and families. FGR is the failure of the fetus to reach its genetically predetermined growth potential. FGR represents a very serious pregnancy complication and, as mentioned above, is the greatest risk factor for stillbirth, a devastating tragedy affecting australian pregnancy 1: 130. In addition, about 27,000 FGR or small for gestational age (SGA; birth weight < 10 th percentile (centile)) infants are born annually in Australia. FGR and SGA at birth are related results of placental insufficiency.

FGR is also a major determinant of perinatal morbidity with low birth weight infants experiencing poor neurological development outcomes (Miller et al (2016) The Journal of Physiology 594: 807-823). Furthermore, FGR is associated with adverse consequences in later life: growth-restricted school-age children have a higher rate of impaired cognition, memory, attention, and motor capacity (Miller et al, (2016) supra). Its effect can last for a lifetime-adults have a higher prevalence of major chronic diseases such as cardiovascular Disease, stroke and diabetes (Barker and Osmond (1986) Lancet 1: 1077-. Placental insufficiency can lead to chronic hypoxemia and reduced nutrient supply to the fetus, which ultimately leads to reduced fetal growth and poor brain development, which can itself lead to neurological problems. These are characteristic of FGR and SGA infants.

Placental insufficiency occurs when the placenta fails to provide adequate oxygen and nutrient exchange for the fetus (Mifsud and Sebire (2014) Total diagnosis and therapy 36: 117-. The fetus responds by: reduce its growth and reallocate resources to ensure survival; blood is delivered to vital organs such as the brain. When these survival adaptations fail, stillbirths occur. There is a need to be able to reliably detect poor fetal growth and placental function so that delivery can occur in time before stillbirth occurs. One major obstacle to reducing the health burden of FGR is the inability to accurately identify SGA fetuses, with FGR cases being more common.

Current methods of detecting FGR are surprisingly poor. The most common screening test for FGR is the detection of SGA fetuses in pregnancy using a tape measure. The maternal abdomen was measured to assess the size of the uterus and if the fetus was suspected to be small, ultrasound was performed. However, this method has a sensitivity (i.e., detection rate) for detecting SGA of only about 20% in normal weight women, and even lower in overweight or obese women. It is expected that Ultrasound delivery to all women in the late stages of pregnancy may reliably pick out all small babies, but this method has been shown to have a detection rate for SGA at birth of only 46% (Fadigas et al (2015) Ultrasound in obstertics and genetics 45:559-565) -57% (Sovio et al (2015) Lancet 386:2089-2097), even in ideal research environments (Fadigas et al (2015) supra). The cost and accessibility of widespread ultrasound has made widespread adoption impractical, highlighting the clear need for new tools to better identify pregnancies affected by FGR. Ultrasound remains a valuable tool in pregnancy management such as the detection of large fetuses (total macrosomia). A giant fetus is a condition in which a newborn has a birth weight significantly greater than the average (i.e., a body weight typically in excess of 4kg), which can lead to birth complications and increase the risk of neonatal injury.

SUMMARY

An assay for monitoring placental health is taught herein that is capable of determining the level or status of placental insufficiency. This information is crucial for successful clinical pregnancy management in order to detect FGR and SGA infants. The ability to reliably detect such conditions, including placental insufficiency, can enable clinical interventions such as close fetal monitoring and early delivery. The present invention teaches that the biomarkers SPINT1 and SYNDECAN1, individually or collectively, are markers of placental health status. Low levels of either or both are indicative of placental insufficiency. Elevated levels of either or both biomarkers may also be an indicator of a condition that adversely affects the fetus, such as a giant fetus.

Thus, achieved herein is an assay for determining placental health status in a female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in the ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SPINT-1 and/or SYNDECAN-1 is a measure of placental sufficiency or a measure of improvement in placental sufficiency.

Also embodied herein is a clinical management regimen for a pregnant mammalian subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein a decrease in the SPINT1 and/or SYNDECAN-1 over time as compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 and/or SYNDECAN-1 is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

Thus, achieved herein is an assay for determining placental health status in a female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in the ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SPINT-1 and/or SYNDECAN-1 is a measure of placental function sufficiency or a measure of improvement in placental function sufficiency.

Also taught herein is an assay for detecting abnormal fetal weight, such as a giant (macromia), in a female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein an increase in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or an increase over time or a change in the ratio relative to a control or a change over time is indicative of a potential giant.

Also taught herein is a clinical management regimen for a neonate in a pregnant mammalian subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein an increase in the SPINT1 and/or SYNDECAN-1 over time as compared to a control or statistically validated level or a change in the rate reflecting an increase in the SPINT1 and/or SYNDECAN-1 level is indicative of a neonate, wherein the fetus is monitored or undergoes early childbirth.

Determination of the concentration or level of a biomarker enables the establishment of a diagnostic rule (diagnostic rule) based on the concentration relative to a control. Alternatively, the diagnostic rules are based on the application of statistical and machine learning algorithms or analytical functions or analytical methods or other data processing means. Such algorithms or analytical functions or analytical methods or other data processing means use the relationship between biomarkers and placental adequacy states observed in training data (with known placental adequacy states) to infer relationships that are subsequently used to predict the state of a patient with an unknown state. An algorithm or analytical function or analytical method or other data processing means providing an index of probability (index of probability) that the patient suffers from placental insufficiency may be used. Algorithms or analytical functions or analytical methods or other data processing means may perform multivariate or univariate analytical functions or other statistical operations.

Thus, in one embodiment, the invention provides diagnostic rules based on the application of statistical and machine learning algorithms or analytical functions or analytical methods or other data processing means. Such algorithms or analytical functions or analytical methods or other data processing means use the relationship between biomarkers and states of placental sufficiency or insufficiency observed in training data (with known placental sufficiency states) to infer relationships that are subsequently used to predict the state of patients with unknown placental sufficiency states. Those skilled in the art of data analysis recognize that many different forms of inference of relationships in training data may be used without materially altering the present invention.

The present invention also contemplates a panel of biomarkers (panel) for detecting placental insufficiency or placental health status or dysfunction in a subject, the panel comprises agents (agents) that specifically bind to the biomarkers to determine the levels of the biomarkers and then optionally subjecting the levels to algorithms or analytical functions or analytical methods or other data processing means generated from a first knowledge base of data, the biomarker is selected from SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1 and at least one other biomarker, the first data repository includes levels of the same biomarker from subjects having a known status with respect to a condition, wherein the algorithm or analytical function or analytical method or other data processing means provides an index of probability that the subject has or does not have placental insufficiency.

Assays taught herein include determining the level of SPINT1, SYNDECAN-1, SPINT1, and SYNDECAN-1 and/or determining the level of SPINT1 and/or SYNDECAN-1 and at least one other biomarker.

Reference to at least one other biomarker includes, but is not limited to, one or more of the following: placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt), and/or Vascular Endothelial Growth Factor (VEGF) as well as other biomarkers and/or placental insufficiency or a result thereof such as a physiochemical parameter of FGR (e.g., ultrasound data).

In one embodiment, the mammalian subject is a human female. However, the present invention extends to veterinary applications. Typically, circulating SPINT1 and/or SYNDECAN-1 is determined in whole blood, plasma or serum. Other fluids may also be tested, including ascites, lymph or urine.

In one embodiment, where the subject is determined to have placental insufficiency, the pregnancy is clinically managed to maximize the health outcome of the fetus. This includes early delivery of the fetus. Similarly, early delivery of the infant may be indicated if it is indicated that the large fetus is due to an elevation in SPINT1 and/or SYNDECAN-1 with or without ultrasound or other physiochemical testing.

The assays of the invention extend to the detection of placental insufficiency or its consequences such as biomarker proteins of FGR or via direct or indirect detection of RNA encoding biomarkers and/or circulating RNA markers.

Although in one embodiment the invention relates to monitoring a decrease in biomarker levels as an indicator of placental insufficiency, detection of elevated levels of SPINT1 and/or SYNDECAN-1 is used as an indicator or predictor of a large fetus and forms part of the invention.

Table 1 provides a list of abbreviations used throughout this specification.

TABLE 1

Abbreviations

Brief Description of Drawings

Some of the figures contain color representations or entities. Color photographs may be obtained on request from the patentee or from the appropriate patent office. If obtained from the patent office, a fee may be charged.

Fig. 1A to 1P are graphical representations showing circulating SPINT1 and SYNDECAN-1 in plasma measured using a commercially available ELISA. SPINT1 was from Sigma Aldrich and SYNDECAN-1 was from Thermo Fisher Scientific. sFLT-1 and PlGF were measured using a diagnostic test assay from Roche. The case control cohort was selected from patients who provided blood samples at 36 weeks gestation. A significant reduction in the level of SPINT1(n 210 control, n 104 SGA) in patients delivering SGA infants (birth weight < 10 th percentile relative to gestational age) (a) with a subject operating area under the curve (AUC) (B) of 0.75. When SYNDECAN-1 was measured in a case control cohort (n 99 controls, n 89 SGAs), a significant reduction of SYNDECAN-1 was also found in women subsequently delivered SGA infants (C), with an auc (d) of 0.73. In contrast, circulating sFLT-1(n 207 controls, n 102 SGA) was significantly increased in women who delivered SGA infants (E) with an auc (f) of 0.58, while circulating PlGF (n 210 controls, 104 SGA) was significantly decreased (G) with an auc (h) of 0.66. Subsequently, the inventors continued to validate the results from the case control cohort in a sample set consisting of 1004 samples collected at 36 weeks gestation. 920 controls and 84 cases where the mother subsequently delivered SGA infants. The inventors found that circulating sprit 1 was significantly reduced in women who subsequently delivered SGA infants (I), with an auc (j) of 0.74. Similarly, SYNDECAN-1 was significantly reduced (K) in women delivered SGA infants, with an auc (l) of 0.65. The time at which these proteins in maternal circulation may be reduced was then assessed for how early in pregnancy. To this end, a case control cohort was selected and circulating SPINT1 (n: 130 controls, n: 104 SGAs) and SYNDECAN-1 (n: 100 controls, n: 84 SGAs) were measured at 28 weeks gestation. At 28 weeks gestation, SPINT1 in women who subsequently delivered SGA infants had been significantly reduced (M), with an auc (n) of 0.69. Similarly, SYNDECAN-1 decreased significantly (O) at 28 weeks gestation, with an auc (p) of 0.69. Data are presented as mean +/-SEM-each symbol represents individual patient p <0.05, p < 0.0001.

Fig. 2A to 2D are graphical representations showing the birth weight percentile for each study infant, which is customized according to maternal birth weight, scheduled weight, height, infant gender and exact gestational age in days. To determine whether there is a relationship between birth weight and circulating SPINT1 levels, two data sets were plotted. Figure 2A shows that the circulating maternal SPINT1 at 36 weeks gestation correlates with the subsequent infant birth percentile — this correlation is plotted in the B-plot for each of the 1004 patients, where there is a statistically significant correlation. Panel C shows that circulating maternal SPINT1(n ═ 145) at 36 weeks gestation was significantly correlated with neonatal lean body mass (lean mass) measured within days after birth. Panel D shows that although SPINT1 at 36 weeks is associated with neonatal lean body mass, it is not associated with the percent skin fold body fat (n ═ 145), also measured within days after birth.

Fig. 3A-3P are graphical representations showing an observational study to determine whether placental SPINT1 has changed in human SGA cases. To this end, a cohort of placentas was selected from controls, preeclampsia or SGA pregnancies. The SPINT1mRNA was measured using quantitative RT-PCR and showed a significant reduction in SPINT1mRNA in both PE and SGA cohorts compared to controls in patients who delivered their infants <34 weeks gestation (a). No change in SPINT1mRNA expression was detected in the placenta collected from patients delivered at >34 weeks gestation (B). Protein expression of the SPINT1 was assessed using western blot and commercially available antibodies (C, D). In samples collected both at <34 weeks of gestation (C) and >34 weeks of gestation (D), the SPINT1 protein in the placenta of women from SGA delivered infants was significantly reduced. Growth limitation is often associated with placental insufficiency and poor placental oxygen supply (leading to placental hypoxia). The inventors evaluated whether exposure of primary placental cells (cytotrophoblast cells isolated from full-term human placenta) to hypoxic conditions (1% oxygen for hypoxia and 8% oxygen for normoxia) would alter SPINT1mRNA and protein expression and protein secretion. The present inventors initially assessed mRNA expression by qRT-PCR (E), and found that exposure of primary placental cells to hypoxia resulted in significantly reduced SPINT1mRNA expression, and a similar finding was observed for cellular proteins measured by western blot (F). Secretion of SPINT1 into the medium bathing the placental cells was measured using the same ELISA as used to measure plasma SPINT1 in blood, and it was found that when the placental cells were made hypoxic, the level of SPINT1 in the medium was also significantly reduced (G). Thus, strong evidence was found to demonstrate that placental hypoxia reduces SPINT 1. In view of the lack of evidence in previous mice that SPINT1 might be associated with placental dysplasia, the present inventors next set out to evaluate whether silencing of SPINT1 using siRNA knockdown in HTR8 placental cell line would affect cell proliferation assessed in real time using the xcelligene system. Indeed, when the SPINT1 in HTR8 cells was silenced, impaired proliferation was found (N). Next, the inventors wanted to know whether this effect on proliferation could be the result of enhanced apoptosis. To determine this, the SPINT1 was silenced in HTR8 cells, and then the proteins were collected and the apoptosis markers BAX, BCL2 and cleaved caspase 3 were measured by western blot. Although the SPINT1 protein expression was found to be significantly reduced as expected (the upper panel shows the lack of bands under sipint), there was no significant change in expression of apoptosis markers, suggesting that reduced proliferation when SPINT1 was silenced was not the result of increased apoptosis (O). Beta-actin was used as loading control. Next, an attempt was made to assess whether augmenting SPINT1 would alter HTR8 placental cell line proliferation. To this end, a commercially available SPINT1 mimetic (Glixx laboratories) called SRI31215 was applied to HTR8 cells and their proliferation was monitored using the xcelligene system. In fact, 5uM or 10uM SRI31215 was found to enhance HTR8 proliferation (P). Data are presented as mean +/-SEM-each symbol represents individual patient or mouse p <0.05, p <0.01, p <0.001, p < 0.0001.

Fig. 4A to 4C are graphical representations (a) showing the increase of the cycle SYNDECAN-1 with the baby birth percentile. The parent SYNDECAN-1 was significantly associated with neonatal lean body mass (B) and percent skinfold body fat (C) measured several days after birth.

Fig. 5A-5E are graphical representations showing an observation study to determine whether placenta SYNDECAN-1 changes in human SGA cases. To this end, a cohort of placentas (same cohort used to measure SPINT 1) was selected from controls, preeclampsia or SGA pregnancies. SYNDECAN-1mRNA was measured using quantitative RT-PCR and showed a significant increase in placental SYNDECAN-1mRNA in both Preeclampsia (PE) and Small for Gestation Age (SGA) cohorts compared to controls in patients who delivered their infants <34 weeks gestation (a). Protein expression was then measured. Commercial antibodies used for western blotting yielded 3 different bands that could correspond to different isoforms of SYNDECAN-1. Densitometric analysis (densitometric analysis) of these bands revealed that in SGA placenta, the 85kDa (b) and 80kDa (c) bands were unchanged, while the 33kDa band was significantly reduced (D). SYNDECAN-1mRNA expression was subsequently measured in the placenta delivered at >34 weeks gestation and no significant change in expression was found (E). Data are presented as mean values +/-SEM-each symbol represents individual patient p <0.05, p <0.01, p <0.001, p < 0.0001.

Fig. 6A-6F are schematic representations of functional studies to determine whether placental SYNDECAN-1 is altered by hypoxia and whether silencing its expression alters cell proliferation. Panels a, B and C show that SYNDECAN-1mrna (a) is significantly reduced in primary human trophoblasts under hypoxia, while cellular proteins are not different (B). Secreted SYNDECAN-1 protein was significantly reduced under hypoxia (C). In pregnant mice exposed to hypoxia, murine SYNDECAN-1 within the placenta was unchanged (D, E). When SYNDECAN-1 was silenced in cells of placental cell line HTR8 using siRNA, cell proliferation was impaired (F). Data are presented as mean +/-SEM-each symbol represents individual patient p <0.05, p < 0.01.

Fig. 7a to 7e show that plasma SPINT1 at 36 weeks is associated with a clinical marker of placental insufficiency. Plasma SPINT1 concentrations at 36 weeks gestation were correlated with Uterine Artery (UA) doppler flow resistance (a), neonatal lean body mass (b), and placental weight (c). The plasma concentration of SPINT1 was gradually reduced in women whose infants were subsequently born with birth weights below the 10 th percentile (d). Plasma SPINT1 concentrations at 36 weeks correlated with birth percentiles (e). The SPINT1 MoMs for both queue 1 and queue 2 are merged and grouped according to the birth percentile. Graphs D and E depict median +/-quartile range. Each individual symbol (c-e) represents a patient. P <0.0001, relative to control (Mann-Whitney).

Fig. 8a to 8d show that the level of SPINT1 is not correlated with the umbilical artery resistance, and with the infant's percent body fat or fat mass (fat mass). The postnatal infant body composition of the sub-cohorts of the entire FLAG cohort was evaluated using a PEAPOD air displacement plethysmography device (air displacement plethyosygraphy device). There was no relationship between circulating SPINT1 levels at 36 weeks and umbilical artery pulsatility index (a), percent body fat (B), or amount of fat (C). The concentration of SPINT1 in plasma and serum samples from women who delivered < 10 th percentile infants at <34 weeks was measured and compared to the concentration of SPINT1 in plasma and serum samples from healthy controls (plasma and serum samples were obtained at the same blood draw). The data show that although SPINT1 was significantly reduced in serum of women with SGA, the degree of change was much less than that observed in plasma. Individual symbols represent individual patients. P <0.0001, p < 0.05.

Fig. 9a to 9j show a comparison of the strength of correlation between SPINT1 or PlGF and clinical markers of placental insufficiency. At 36 weeks, plasma spinnt 1 concentrations appeared to have a stronger correlation with birth weight percentile (a vs f), placenta weight (b vs g), lean body mass (c vs h), and uterine artery resistance (d vs i) compared to PlGF. Both molecules are independent of the umbilical artery resistance (e and j). Individual symbols represent individual patients.

Fig. 10A-10H show the correlation between SPINT1 or SYNDECAN-1 and various clinical parameters of placental insufficiency in a larger sample cohort obtained from up to 2040 women. Each symbol represents an individual patient. D plot, data are expressed as median +/-quartile range. P < 0.0001.

Fig. 11A-11C demonstrate the change in SPINT1 at 28 weeks in the entire cohort of n-2040 (n-1827 controls, n-213 cases). The data demonstrate a significant reduction in circulating SPINT1 at 28 weeks in women delivering SGA infants (a), with an auc (b) of 0.60. At 28 weeks gestation, SPINT1 was consistently associated with the birth weight percentile (C). Each symbol represents an individual patient. P < 0.0001.

Fig. 12A-12D demonstrate the change in plasma SPINT1 at 36 weeks in the larger cohort and show a correlation with markers of placental insufficiency. Plasma SPINT1 concentrations at 36 weeks gestation were correlated with Uterine Artery (UA) doppler flow resistance (a, n ═ 325), neonatal lean body mass (B, n ═ 281), and placental weight (C, n ═ 378). The plasma concentration of SPINT1 was gradually reduced in women whose infants were subsequently born with birth weights below the 10 th percentile (D). Plot D depicts median +/-quartile range. Each individual symbol (a-D) represents a patient. P <0.0001, relative to control.

Figures 13A to 13D show SPINT1 and SYNDECAN-1 measured in a separate cohort of 556 samples collected at the day of delivery (caesarean section) or induction of labor at the Mercy Hospital for Women (department of labor) of melbourne, australia. In this cohort, we demonstrated that also SPINT1(A, B) was significantly reduced in women carrying SGA infants, with an AUC of 0.66 (n-47 SGA, n-509 control). Similarly, SYNDECAN-1 was also significantly reduced in women carrying SGA infants in this cohort (C, D), with an AUC of 0.644.

Detailed description of the invention

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

As used in this specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a single biomarker as well as two or more biomarkers; reference to "an assay" includes a single assay as well as two or more assays; reference to "the (the) disclosure" includes a single aspect as well as more than one aspect taught by the disclosure; and so on. The term "invention" encompasses the various aspects taught and embodied herein. The "forms" of the invention encompass any of the variants and derivatives contemplated herein. All aspects and forms of the invention are realized within the scope of the claims.

The use of numerical values in the various ranges specified in this application are stated as approximations, as both the minimum and maximum values within the stated ranges are prefaced by the word "about," unless expressly stated otherwise. In this manner, minor variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Further, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. Furthermore, the present invention extends to the ratio of two or more markers, providing a numerical value that correlates with the level of risk of placental insufficiency.

The present invention relates to an assay for determining the circulating level of one or either or both of the biomarker SPINT1 and/or the biomarker SYNDECAN-1 in a pregnant mammalian subject. The SPINT1 and/or syncean-1 can be determined separately or together, either alone or in combination with one or more other biomarkers and/or one or more physiochemical parameters (such as data from ultrasound). These biomarkers, when present at reduced levels, are considered indicators of placental insufficiency. These biomarkers, when elevated, are considered to be indicative of fetal weight abnormalities, including but not limited to giant fetuses, alone or in combination with one or more biomarkers and one or more physiochemical parameters (such as data from ultrasound).

The term "determining" includes, but is not limited to, a method, protocol, step, or series of steps and/or processes for determining the level or turnover (velocity) of the SPINT1 and/or SYNDECAN-1. The level of SPINT1 and/or SYNDECAN-1 can be compared to a standardized control, or the level of SPINT1 and/or SYNDECAN-1 can be compared to a statistically validated predetermined level. An example of a standardized control is the level in a normal pregnant female subject of the same age and physical characteristics. The turnover rate includes the rate or extent of increase or decrease in the level of any biomarker. The level or turnover rate of the SPINT1 and/or SYNDECAN-1 can be expressed as a concentration level in, but is not limited to, an amount of picograms, nanograms, micrograms, or milligrams per volume of circulating fluid (typically in milliliters, although it can be expressed in any volumetric amount). Optionally, the levels or turnover rates of the biomarkers are physicochemically in the form of ratios between each other and/or between one of the two and another biomarker, such as placental growth factor (PlGF) or other marker, soluble fms-like tyrosine kinase-1 (sFlt) or Vascular Endothelial Growth Factor (VEGF).

The level or turnover rate of SPINT1 and/or SYNDECAN-1 can also be analyzed by, but not limited to, multivariate or univariate algorithms or other analytical functions to establish a value that is compared to a control or statistically validated predetermined level.

In essence, the present invention teaches that a decrease in the level or turnover of SPINT1 and/or SYNDECAN-1 in a pregnant female mammalian subject is indicative of placental insufficiency. Depending on what the clinical intervention initiated is, the level of SPINT1 and/or SYNDECAN-1 may be monitored throughout the pregnancy and/or measured at any time during a predetermined period including a first trimester, second trimester or third trimester period. For the avoidance of doubt, the determination may be made at any pregnancy time point or time window throughout the pregnancy period. The ability to detect placental insufficiency via a biomarker or combination of biomarkers improves sensitivity and specificity to levels well above physical measurements, ultrasound, examination, and/or birth weight or predicted fetal weight. These are all encompassed by the term "physiochemical parameters".

Thus, achieved herein is an assay for determining placental health status in a female mammalian subject, the method comprising determining a circulating level of SPINT1 and/or SYNDECAN-1, wherein a decrease in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in the ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SPINT-1 and/or SYNDECAN-1 is a measure of adequate placental function or a measure of improvement in adequate placental function.

As used herein, the term "indicative" (e.g., indicative of placental insufficiency or indicative of placental sufficiency) refers to a sign or indication or factor to be considered, rather than explicit evidence of itself, and generally refers to an increased likelihood of the presence of a particular condition. For example, a decrease or decrease in the level or concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a change in the ratio relative to a control or a change over time is generally associated with an increased likelihood of placental insufficiency. Likewise, an increase in the level or concentration of SPINT1 and/or SYNDECAN-1 relative to a control or an increase over time or a change in the ratio relative to a control or a change over time is generally associated with an increased likelihood that placental function is sufficient.

In one embodiment, the assay determines placental health in a female mammalian subject, the method comprising determining a circulating level of SPINT1, wherein a decrease in concentration of SPINT1 relative to a control or a decrease over time or a change in ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SPINT-1 is a measure of adequate placental function or a measure of improvement in adequate placental function.

In one embodiment, the assay determines placental health status in a female mammalian subject, the method comprising determining a circulating level of SYNDECAN-1, wherein a decrease in concentration of SYNDECAN-1 relative to a control or a decrease over time or a change in ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SYNDECAN-1 is a measure of adequate placenta function or a measure of improvement in adequate placenta function.

In one embodiment, the assay determines placental health status in a female mammalian subject, the method comprising determining circulating levels of SPINT1 and at least one other biomarker, wherein a decrease in concentration of SPINT1 relative to a control or decrease over time and a change in at least one other biomarker relative to a control or change over time or a change in ratio relative to a control or change over time indicates placental insufficiency, and an increase in SPINT-1 and a change in at least one other biomarker is a measure of adequate placenta function or a measure of improvement in adequate placenta function.

In one embodiment, the assay determines placental health status in a female mammalian subject, the method comprising determining circulating levels of SYNDECAN-1 and at least one other biomarker, wherein a decrease in concentration of SYNDECAN-1 relative to a control or a decrease over time and a change in at least one other biomarker relative to a control or a change over time or a change in the ratio relative to a control or a change over time indicate placental insufficiency, and an increase in SYNDECAN-1 and a change in at least one other biomarker is a measure of adequate placenta function or a measure of improvement in adequate placenta function.

An assay to determine placental health status in a female mammalian subject is achieved herein, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in concentration or a decrease over time or a change in ratio or a change over time of the SPINT1 and/or SYNDECAN-1 relative to a control is indicative of placental insufficiency, and an increase in SPINT-1 and/or SYNDECAN-1 is a measure of adequate placental function or a measure of improvement in adequate placental function.

Also taught herein is an assay for detecting abnormal fetal weight, such as a giant child, in a female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein an increase in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or an increase over time or a change in the ratio relative to a control or over time is indicative of a potential giant child.

Also embodied herein is an assay to detect abnormal fetal weight in a female mammalian subject, the method comprising determining maternal circulation levels of the SPINT1 and/or SYNDECAN-1, wherein an increase in the concentration of the SPINT1 and/or SYNDECAN-1 relative to a control or an increase in the ratio relative to a control or a change over time is indicative of potentially high fetal weight, and a decrease in the concentration of the SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in the ratio relative to a control or a change over time is indicative of potentially low fetal weight or less fetal age or fetal growth restriction.

The invention extends to any mammalian subject, such as a human, non-human primate, farm animal (e.g. sheep, cattle, horse, pig, alpaca, llama), competitor animal (e.g. horse, camel or greyhound dog) or domestic animal (e.g. dog or cat). Racing equines include quart horses, thoroughbred horses, arabic horses and warm-blooded horses. Thus, the invention has application in human and veterinary clinical practice.

In one embodiment, the mammalian subject is a pregnant human female. In another embodiment, the mammalian subject is a pregnant mare (mare). In yet another embodiment, the mammalian subject is a pregnant mare.

The results of the assay may be used in conjunction with one or more risk factors associated with the subject to further aid in making a diagnosis. The term "risk factor" is meant to include any factor that statistically increases the risk of an abnormal fetal weight. Risk factors associated with abnormal fetal weight include, but are not limited to, maternal age, pre-pregnancy weight index, educational status, smoking, drinking, in vitro fertilization, anemia of pregnancy, pre-eclampsia, diabetes, gestational age, weight gain in pregnancy, neonatal gender, history of large fetus, low fetal weight or history of small fetal age or fetal growth restriction.

Accordingly, taught herein is an assay to determine placental health status in a pregnant human female subject, the method comprising determining a circulating level of SPINT1 and/or SYNDECAN-1, wherein a decrease in concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease in ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SPINT-1 and/or SYNDECAN-1 is a measure of adequate placental function or a measure of improvement in adequate placental function.

In one embodiment, the assay determines placental health status in a pregnant human female subject, the method comprising determining a circulating level of SPINT1, wherein a decrease in concentration of SPINT1 relative to a control or a decrease over time or a change in ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SPINT-1 is a measure of adequate placental function or a measure of improvement in adequate placental function.

In one embodiment, the assay determines placental health status in a pregnant human female subject, the method comprising determining a circulating level of SYNDECAN-1, wherein a decrease in concentration of SYNDECAN-1 relative to a control or a decrease over time or a change in ratio relative to a control or a change over time is indicative of placental insufficiency, and an increase in SYNDECAN-1 is a measure of adequate placenta function or a measure of improvement in adequate placenta function.

In one embodiment, the assay determines placental health status in a pregnant human subject, the method comprising determining circulating levels of SPINT1 and at least one other biomarker, wherein a decrease in concentration of SPINT1 relative to a control or decrease over time and a change in at least one other biomarker relative to a control or change over time or a change in ratio relative to a control or change over time indicates placental insufficiency, and an increase in SPINT-1 and a change in at least one other biomarker is a measure of adequate placenta function or a measure of improvement in adequate placenta function.

In one embodiment, the assay determines placental health status in a pregnant human subject, the method comprising determining circulating levels of SYNDECAN-1 and at least one other biomarker, wherein a decrease in concentration of SYNDECAN-1 relative to a control or a decrease over time and a change in at least one other biomarker relative to a control or a change over time or a change in ratio relative to a control or a change over time indicate placental insufficiency, and an increase in SYNDECAN-1 and a change in at least one other biomarker is a measure of adequate placenta function or a measure of improvement in adequate placenta function.

Also embodied herein is a clinical management regimen for a pregnant human subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein a decrease in the SPINT1 and/or SYNDECAN-1 over time as compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 and/or SYNDECAN-1 is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

Also taught herein is a clinical management regimen for a neonate in a pregnant human subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein an increase in the SPINT1 and/or SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting an increase in the SPINT1 and/or SYNDECAN-1 level is indicative of a neonate, wherein the fetus is monitored and is likely to undergo early delivery.

The term "subject" includes patients, pregnant mothers and females of reproductive age.

Biomarkers in maternal circulating fluid including whole blood, plasma and serum are determined. Other circulating fluids, such as lymph, ascites or urine, may also be assayed. Biomarkers can be determined along with or in combination with physiochemical data, such as obtained by ultrasound.

The assay can be performed in any number of ways, including direct measurement of the biomarker or indirect measurement using a ligand for the biomarker. "ligands" include antibodies and receptors for biomarkers. Conveniently, the assay is an immunoassay such as an ELISA and uses an antibody that specifically binds to SPINT1 or SYNDECAN-1 and detects either directly or indirectly SPINT1 or SYNDECAN-1. The assay may be quantitative. The assay may also be a genetic assay, such as detecting RNA and/or other circulating RNA species (or non-RNA nucleic acid species) encoding biomarkers, which are indicative of placental insufficiency or its associated conditions such as FGR.

Thus, a rapid, efficient and sensitive assay for the identification of placental insufficiency is provided. Conditions of placental insufficiency include results caused directly or indirectly by placental insufficiency, such as FGR and SGA infants. In a particular embodiment, the assay enables early detection of placental insufficiency. Nevertheless, the present invention is not limited to the early detection of placental insufficiency, as the assay can be used for any gestational stage of pregnancy. Furthermore, elevated levels of SPINT1 and/or SYNDECAN-1, or a ratio indicative of one or both of them elevated, are considered indicators of a large fetus.

As described elsewhere herein, the inventors have also surprisingly found that the circulating level of SPINT1 in pregnant female subjects correlates with a clinical parameter of placental insufficiency, such as uterine blood flow (uterine artery doppler velocity) (R) at the time of sampling²＝0.111；p<0.0001), neonatal lean body mass (R)²＝0.064；p<0.0001), placenta weight (R)²＝0.087；p<0.0001) and placental surface area (R)²＝0.028；p<0.013)。

Circulating biomarkers that can be used to detect placental insufficiency or to assess placental health levels are identified. Biomarkers are SPINT1 (also known as hepatocyte growth factor activator inhibitor-1; HAI-1) and SYNDECAN-1. References to "SPINT 1" and "SYNDECAN-1" include modified or homologous forms thereof. Modified forms include derivatives, polymorphic variants, truncated forms (truncates) and aggregated or multimeric forms or forms with expansion elements (e.g. amino acid expansion elements). For the sake of brevity, such modified forms and homologue forms are included by reference to any biomarker or some or all biomarkers.

In one embodiment, the SPINT1 comprises the amino acid sequence of SEQ ID NO:1(GenBank accession No. AB000095.1), or an amino acid sequence having at least 70% sequence identity thereto:

in one embodiment, SYNDECAN-1 comprises the amino acid sequence of SEQ ID NO:2(GenBank accession number: AJ551176.1), or an amino acid sequence having at least 70% sequence identity thereto:

modified or homologous forms of SPINT1 are familiar to those skilled in the art, illustrative examples of which are described in GenBank accession numbers: NM _001032367.1 to NP _001027539.1 (isoform 2 precursor), NM _003710.3 to NP _003701.1 (isoform 2 precursor), and NM _181642.2 to NP _857593.1 (isoform 1 precursor), the contents of which are incorporated herein by reference in their entirety.

Modified or homologous forms of SYNDECAN-1 are also familiar to those skilled in the art, illustrative examples of which are described in GenBank accession numbers: XM _005262622.2 to XP _005262679.1 (isoform X3), GenBank accession No.: XM _005262620.4 to XP _005262677.1 (isoform X1) and GenBank accession numbers: XM _005262621.3 to XP _005262678.3 (isoform X2), the contents of which are incorporated herein by reference in their entirety. Additional illustrative examples of modified or homolog forms of SYNDECAN-1 are described in Romaris et al (1999, The Journal of Biological Chemistry,274,18667-18674), The contents of which are incorporated herein by reference in their entirety.

Reference to "at least 70% sequence identity" includes 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, for example, after optimal alignment or optimal match analysis. Thus, in one embodiment, the amino acid sequence of the SPINT1 comprises at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or more preferably 100% sequence identity to SEQ ID No. 1 as described herein. In another embodiment, the amino acid sequence of SYNDECAN-1 comprises at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% or more preferably 100% sequence identity to SEQ ID No. 2 as described herein.

The terms "identity," "similarity," "sequence identity," "sequence similarity," "homology," "sequence homology" and the like as used herein mean the identity of amino acid residues between aligned sequences at any particular amino acid residue position in the aligned sequences. The term "similarity" or "sequence similarity" as used herein indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are of a similar type. For example, leucine may be substituted for an isoleucine or valine residue. As described elsewhere herein, this may be referred to as a conservative substitution. In one embodiment, the modified or homologous forms of SPINT1 and SYNDECAN-1 have amino acid sequences that differ from SEQ ID NOs: 1 and 2, respectively, by way of one or more conservative substitutions of any amino acid residue contained therein.

In some embodiments, sequence identity with respect to a peptide sequence relates to the percentage of amino acid residues in the candidate sequence that are identical to residues in the corresponding peptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent homology and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions, nor insertions should be construed to reduce sequence identity or homology. Methods and computer programs for aligning two or more amino acid sequences and determining their sequence identity or homology are well known to those skilled in the art. For example, the percent identity or similarity of two amino acid sequences can be readily calculated using algorithms such as the BLAST, FASTA or Smith-Waterman algorithm. Techniques for determining amino acid sequence "similarity" are well known to those skilled in the art. In general, "similarity" refers to the precise comparison of amino acids to amino acids at appropriate positions of two or more peptide sequences, where the amino acids are identical or have similar chemical and/or physical properties, such as charge or hydrophobicity. The so-called "percent similarity" between the compared peptide sequences can then be determined. In general, "identity" refers to the precise amino acid-to-amino acid correspondence of two peptide sequences. Two or more peptide sequences may also be compared by determining their "percent identity". The percent identity of two sequences can be described as the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. The local homology algorithm of Smith and Waterman, Advances in Applied Mathesics 2:482-489(1981) provides an approximate alignment of nucleic acid sequences. The algorithm can be extended to the use of scoring matrices developed by Dalhoff (Atlas of Protein Sequences and structures, M.O.Dalhoff ed.,5 supl.3: 353-. Suitable procedures for calculating percent identity or similarity between sequences are well known in the art. Optimal alignment of sequences for alignment of the comparison window (compare window) can be performed by computerized implementation of an algorithm (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, pub. 7.0, Genetics Computer Group,575Science Drive Madison, Wis., USA) or by inspection and optimal alignment results from any of a variety of methods of choice (i.e., results in the highest percentage homology of the comparison window). Reference may also be made to the BLAST program family as disclosed, for example, by Altschul et al, (1997, nucleic acids Res.25: 3389). A detailed discussion of sequence analysis can be found in Ausubel et al ("Current Protocols in Molecular Biology", John Wiley & Sons Inc,1994-1998, Chapter 15) at unit 19.3.

As described herein, modified or homologous forms of SPINT1 and SYNDECAN-1 include non-human isoforms. Illustrative examples of non-human isotypes include the natural SPINT1 and SYNDECAN-1 isotypes of primates, companion animals such as cats and dogs, working animals (working animals) such as horses, donkeys, etc., farm animals such as sheep, cows, goats, pigs, etc., laboratory test animals such as rabbits, mice, rats, guinea pigs, hamsters, etc., and captive wild animals such as wild animals in zoos and wilderness, deer, wild dogs, etc.

Thus, another aspect achieved herein is an assay for determining placental functional sufficiency status in a subject, the assay comprising determining the concentration of a biomarker selected from the group consisting of SPINT1 and SYNDECAN-1 in a circulating biological sample from the subject, wherein a change in the level of the biomarker relative to a control provides an indication of placental functional sufficiency status. In one embodiment, a decrease in SPINT1 and/or SYNDECAN-1 is indicative of placental insufficiency or a propensity to develop placental insufficiency.

In an alternative embodiment, the present invention contemplates an assay for determining the state of placental functional sufficiency in a subject, the assay comprising determining the concentration of a biomarker selected from the group consisting of SPINT1 and SYNDECAN-1 in a circulating biological sample from the subject; subjecting the levels to an algorithm or analytical function or analytical method or other data processing means generated from a first knowledge base of data comprising levels of the same biomarkers from subjects or cohorts of subjects having a known status with respect to sufficiency of placental function, wherein the algorithm or analytical or data processing means provides an index of probability that a subject has or does not have placental insufficiency or placental sufficiency. Reference to an "algorithm" includes an algorithm that performs, but is not limited to, a multivariate or univariate analytical function.

The above aspects are also applicable to detecting or monitoring giant fetuses by screening for elevated levels of SPINT1 and/or SYDNECAN-1.

Determination of the concentration or level of the biomarker enables the establishment of a diagnostic rule based on the concentration relative to a control. Optionally, the diagnostic rules are based on the application of statistical and machine learning algorithms. Such algorithms use the relationship between biomarkers and placental adequacy states observed in training data (with known placental adequacy states) to infer relationships that are subsequently used to predict the state of a patient with an unknown state. An algorithm may be used that provides an index of the probability that a patient has placental insufficiency. As described above, the algorithm may perform, but is not limited to, multivariate or univariate analysis functions. Optionally, the data is subjected to an analysis or analysis function or process.

Thus, in one embodiment, the present invention provides diagnostic rules based on the application of statistical and machine learning algorithms. Such algorithms use the relationship between biomarkers and states of placental insufficiency or sufficiency observed in training data (with known placental adequacy states) to infer relationships that are subsequently used to predict the state of patients with unknown placental adequacy states. Those skilled in the art of data analysis recognize that many different forms of inference of relationships in training data may be used without materially altering the present invention. Other analytical methods may also be employed to analyze the data and identify correlations between biomarkers and/or physiochemical parameters and placental insufficiency or conditions associated therewith, such as FGR.

Thus, the present invention contemplates the use of a training data knowledge base generating algorithm or analysis function or analysis method or other data processing means, the training data knowledge base comprising biomarker levels selected from the group consisting of: (ii) SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1 and at least one other biomarker, said algorithm or analytical function or analytical method or other data processing means providing a probability index predicting a property of the placenta after inputting a second database comprising levels of the same biomarker from patients with unknown placental functional sufficiency states. Other analysis functions may also be used.

As noted above, a "subject" is typically a human female. However, the present invention extends to veterinary applications.

The term "training data" includes knowledge of the levels of biomarkers relative to controls. "control" includes comparison to biomarker levels in subjects with appropriate placental sufficiency or with known placental insufficiency, or may be statistically determined levels based on the test. The statistically determined level may be a statistically verified predetermined level or cutoff value that has been verified as being associated or correlated with adequate placental function or placental insufficiency. The term "level" also encompasses the ratio of biomarker levels and their turnover rates.

"training data" also includes the concentration of one or more of SPINT1 and/or SYNDECAN-1. The data may include information about an increase or decrease in one or both of the biomarkers.

The present invention also contemplates a panel of biomarkers for detecting placental insufficiency or placental health state or dysfunction in a subject, the panel comprising agents that specifically bind to the biomarkers to determine the levels of the biomarkers and then optionally subjecting the levels to an algorithm or analytical function or analytical method or other data processing means generated from a first data repository selected from the group consisting of SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1 and at least one other biomarker, said first data repository comprising levels of the same biomarkers from subjects having a known state with respect to a condition, wherein the algorithm provides a probability index that the subject has or does not have placental insufficiency.

The level or concentration of the biomarker (SPINT1 and/or SYNDECAN-1) provides input test data referred to herein as a "second data knowledge library". The second data knowledge base is considered relative to a control or is input into an algorithm generated from a "first data knowledge base" that includes information on biomarker levels in subjects with known placental sufficiency or placental insufficiency. The second data repository is from subjects with unknown status regarding placental sufficiency or placental insufficiency. The output of the algorithm or analytical function or analytical method or other data processing means is a probability or risk factor (referred to herein as a probability index) that the subject has a particular level of adequate or insufficient placental function. Algorithms or analytical functions or analytical methods or other data processing means may perform multivariate or univariate analytical functions or other statistical operations.

Agents that "specifically bind" to a biomarker typically include immunointeractive molecules (immunointeractive molecules), such as antibodies or hybrids (hybrids), derivatives, including recombinant or modified forms thereof or antigen binding fragments thereof. The agent may also be a receptor or other ligand. The antibodies or receptors may be specific for biomarkers from a particular mammalian species, or may be produced from different species if they cross-react. All these agents help to determine the level of the biomarker. The information about the level is input data to the algorithm.

Thus, the invention also provides a panel of immobilized ligands against either SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1 with at least one other biomarker.

Ligands, such as biomarker-specific antibodies, can quantitatively or qualitatively detect or determine the level of the biomarker. Reference to "levels" includes concentrations, as well as ratios and turnover rates of levels, by weight/volume, activity/volume or units/volume or other convenient representation.

As mentioned above, a "sample" is typically a circulating maternal fluid, such as whole blood, plasma or serum. Alternatively, the sample is a tissue sample that is being subjected to a histological examination, or is ascites, lymph fluid or urine.

As noted above, terms such as "ligand" or "binding agent" refer to any compound, composition, or molecule capable of specifically or substantially specifically (i.e., with limited cross-reactivity) binding to an epitope on SPINT1 or SYNDECAN-1. "binding agents" typically have a single specificity. Nevertheless, binding agents with multiple specificities for both SPINT1 and SYNDECAN-1 are also contemplated herein. The binding agent (or ligand) is typically an antibody, such as a monoclonal antibody or derivative or analog thereof, but also includes, but is not limited to: (iv) an Fv fragment; a single chain fv (scFv) fragment; a Fab' fragment; a F (ab') 2 fragment; humanized antibodies and antibody fragments; camelized antibodies and antibody fragments; and multivalent forms of the foregoing. If appropriate, multivalent binding agents may also be used, including but not limited to: monospecific or bispecific antibodies; such as disulfide stabilized Fv fragments, scFv concatemers [ (scFv)₂Fragments]Diabody, triabodyAntibodies (tribodies) or tetrabodies (tetrabodies), which are generally covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments. "binding agents" also include aptamers, as described in the art.

Methods for making antigen-specific binding agents, including antibodies and derivatives and analogs thereof, and aptamers are well known in the art. Polyclonal antibodies can be generated by immunizing an animal. Monoclonal antibodies can be prepared according to standard (hybridoma) methods. Antibody derivatives and analogs, including humanized antibodies, can be recombinantly produced by isolating DNA fragments from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. Phage display and aptamer technology is described in the literature and allows for the in vitro clonal amplification of antigen-specific binding reagents with affinity and very low cross-reactivity. Phage display reagents and systems are commercially available and include the Recombinant Phage Antibody System (RPAS) commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, N.J., and the pSKAN phagemid display system commercially available from MoBiTec, LLC of Marco Island, Florida. Aptamer technology is described, for example and without limitation, in U.S. patent nos. 5,270,163; U.S. Pat. No. 5,475,096; nos. 5,840,867 and 6,544,776.

ECLIA, ELISA and Luminex LabMAP immunoassays are examples of suitable assays for detecting biomarker levels. In one example, a first binding reagent/antibody is attached to the surface and a second binding reagent/antibody containing a detectable group is bound to the first antibody. Examples of detectable groups include, for example and without limitation: a fluorescent dye, an enzyme, an epitope for binding to the second binding reagent (e.g., when the second binding reagent/antibody is a mouse antibody detected by a fluorescently labeled anti-mouse antibody), e.g., an antigen or a member of a binding pair, such as biotin. The surface may be a planar surface, such as in the case of typical grid-type arrays (e.g., without limitation, 96-well plates and planar microarrays), or a non-planar surface, such as using a coated bead array technique, in which each "bead" is labeled with, for example, a fluorescent dye (such as the Luminex technique described in U.S. patent nos. 6,599,331, 6,592,822, and 6,268,222) or a quantum dot technique (e.g., as described in U.S. patent No. 6,306,610). Such assays may also be considered as Laboratory Information Management Systems (LIMS).

In bead-type immunoassays, the Luminex LabMAP system can be used. The LabMAP system included polystyrene microspheres internally stained with two fluorochromes having different spectra. Using the exact ratios of these fluorescent dyes, an array consisting of groups of 100 different microspheres with a specific spectral address (spectral address) was created. Each microsphere set may have a different reactant on its surface. Because the sets of microspheres can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to the reporter molecule quantifies the biomolecular interactions that occur on the surface of the microsphere. The microspheres are interrogated individually in a fast flowing fluid stream as they pass through two separate lasers in a Luminex analyzer. High speed digital signal processing classifies the microspheres based on their spectral addresses and quantifies the reaction on the surface for each sample in a few seconds.

As used herein, "immunoassay" refers to an immunoassay (immune assays), typically but not exclusively a sandwich assay, capable of detecting and quantifying a desired biomarker, i.e., either SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1 or one of SPINT1 or SYNDECAN-1, with at least one other biomarker.

Data generated from assays that determine circulating fluid levels of either SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1, or one of SPINT1 or SYNDECAN-1, with at least one other biomarker, can be used to determine the likelihood or progression of a fetal disc insufficiency state in a subject. The data input comprising biomarker levels is compared to a control or input into an algorithm that provides a risk value for the likelihood that the subject has or will develop placental insufficiency.

In the context of the present disclosure, "circulating fluid" includes any blood fraction (fraction), such as serum or plasma, that can be analyzed according to the methods described herein. Measuring the blood level of a particular biomarker means that any suitable blood fraction can be tested to determine the blood level, and the data can be reported as the value present in that fraction. Other fluids contemplated herein include ascites, lymph, and urine.

The invention also encompasses a composition comprising a sample obtained from a pregnant mammalian subject and an antibody that specifically binds to SPINT1 or SYNDECAN-1. In some embodiments, the composition comprises an antibody that specifically binds to SPINT1 and an antibody that specifically binds to SYNDECAN-1. Suitably, the sample is a circulating maternal fluid, such as whole blood, plasma or serum. Alternatively, the sample is a tissue sample that is being subjected to a histological examination, or is ascites, lymph fluid or urine. The one or more antibodies are suitably labeled with a detectable group or substance.

As described above, a method for diagnosing a placental health or dysfunction state is provided by determining the level of a particular biomarker, and using that level as a second data knowledge base in an algorithm generated using the first data knowledge base or the level of the same biomarker in patients with known placental health. Also provided are methods of detecting placental dysfunction, comprising determining the presence and/or turnover rate of a particular identified biomarker in a sample from a subject. By "turnover rate" is meant the change in biomarker concentration over time in a patient's sample (maternal circulating fluid).

In one embodiment, the present invention contemplates a method for monitoring pregnancy progression in a patient, the method comprising:

(a) providing a sample of circulating fluid from a patient;

In one embodiment, an increased index of probability of developing placental dysfunction at a later time point may indicate that the condition is progressing and that the treatment (if applicable) is ineffective. In contrast, a probability index that decreases at a later point in time may indicate that the placental function sufficiency is improving, and that the treatment (if applicable) is effective. Treatment may include premature or early delivery of the infant.

As used herein, the terms "early delivery" and "early delivery" are used interchangeably herein to refer to the delivery of a fetus before a pregnant subject undergoes natural delivery, and include delivery assistance interventions such as induction of labour. The early or early delivery may be preterm (pre-term) (e.g., prior to 37 weeks gestation), term (term) (e.g., 37, 38, 39, 40, 41, or 42 weeks gestation), or expired (post-term) (e.g., after 42 weeks gestation), and includes vaginal delivery, instrumental delivery (forceps or vacuum delivery), or caesarean delivery. In particular embodiments, the terms "preterm labor" and "early delivery" refer to a childbirth assistance intervention (e.g., induction) at 38 weeks, 38.5 weeks, or 39 weeks of gestation.

As noted above, antibodies can be used in any of a number of immunoassays that rely on binding interactions between an antigenic determinant of a biomarker and an antibody. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ECLIA, ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. The antibodies can be used to detect and quantify the levels of biomarkers in a sample to determine the level of placental insufficiency or placental insufficiency.

The antibody or circulating fluid sample may be immobilized on a carrier or solid support capable of immobilizing cells, antibodies, etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamide, gabbros and magnetite. The support material may have any possible configuration, including spherical (e.g., beads), cylindrical (e.g., the inner surface of a test tube or well, or the outer surface of a rod), or flat (e.g., sheet, test strip). An indirect method may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a secondary antibody specific for the antibody reacting with the biomarker protein. For example, if the antibody specific for the biomarker protein is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma globulin labeled with a detectable substance as described herein.

When using a radiolabel as detectable substance, the biomarker can be located by autoradiography. The results of autoradiography can be quantified by determining the density of particles in autoradiography by a variety of optical methods, or by counting particles.

The methods of the invention described herein can also be performed using microarrays such as oligonucleotide arrays, mRNA arrays, cDNA arrays, genomic DNA arrays, or tissue arrays. Any nucleic acid species present in the maternal circulating fluid and directly or indirectly associated with placental insufficiency or a condition associated therewith, such as FGR or SGA infants, can be determined.

In one embodiment, the methods of the invention comprise detecting the expression of a nucleic acid molecule encoding one of SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1, or SPINT1 or SYNDECAN-1, and at least one other biomarker, and determining the level of the biomarker based on the expression level. Alternatively, another nucleic acid species (e.g., an RNA species other than mRNA) directly or indirectly associated with placental insufficiency or a condition associated therewith, such as FGR or SGA infants, can be determined. One skilled in the art can construct nucleotide probes for detecting mRNA sequences or other RNA species encoding biomarkers in a sample. Suitable probes include nucleic acid molecules based on a nucleic acid sequence encoding at least five consecutive amino acids from a biomarker region, preferably they comprise 15 to 30 nucleotides. The nucleotide probe may be labeled with a detectable substance such as a radioactive label that provides sufficient signal and has a sufficient half-life such as³²P、³H、⁴⁴C, and the like. Other detectable substances that may be used include antigens recognized by a specifically labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds. Suitable labels may be selected based on the hybridization and binding rate of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. The labeled probe may be hybridized to nucleic acid on a solid support such as a nitrocellulose filter or a nylon membrane, as generally described in Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd ed.), 1989. Nucleic acid probes can be used to detect genes encoding biomarkers in, for example, human cells. Nucleotide probes may also be used to diagnose placental disorders involving SPINT1 and/or SYNDECAN-1, to monitor the progression of such disorders, or to monitor treatment. In one embodiment, the probes are used to diagnose and monitor the progression of adequate or inadequate placental function.

The probes can be used in hybridization techniques to detect expression of genes encoding biomarker proteins. The techniques generally involve contacting and incubating nucleic acids (e.g., mRNA or other RNA species) obtained from a sample of a patient or other cellular source with a probe under conditions that facilitate specific annealing of the probe to complementary sequences in the nucleic acids. After incubation, non-annealed nucleic acids are removed and the presence of nucleic acids hybridized to the probe (if any) is detected.

Detection of mRNA can include converting mRNA to cDNA and/or amplifying a particular nucleotide sequence using amplification methods such as Polymerase Chain Reaction (PCR), and then analyzing the amplified molecule using techniques known to those skilled in the art. Suitable primers can be routinely designed by those skilled in the art.

The hybridization and amplification techniques described herein can be used to determine qualitative and quantitative aspects of the expression of genes encoding biomarkers. For example, RNA can be isolated from cell types or tissues known to express genes encoding biomarkers or otherwise associated with placental insufficiency, and tested using hybridization (e.g., standard Northern analysis) or PCR techniques as described herein. Techniques are available for detecting transcript size differences that may result from normal or aberrant alternative splicing. Techniques are available for detecting quantitative differences between full-length and/or alternatively spliced transcript levels detected in normal individuals relative to those individuals exhibiting symptoms of placental insufficiency involving biomarker proteins or genes.

Accordingly, the present invention provides a method of detecting placental health in a subject, the method comprising:

(a) providing a sample of circulating fluid from a patient;

(b) extracting nucleic acid molecules from the sample or cells within the sample, the nucleic acid molecules comprising mRNA or other RNA species encoding a biomarker or portion thereof;

(d) determining the level of mRNA or other RNA species encoding a biomarker; and

The methods described herein can be performed by using a pre-packaged diagnostic kit containing the necessary reagents to perform any of the methods of the invention. For example, a kit may comprise at least one specific nucleic acid or antibody described herein, which may be conveniently used, for example, in a clinical setting, to screen and diagnose patients, and to screen and identify those individuals who exhibit a predisposition to develop placental dysfunction. The kit may further comprise nucleic acid primers for amplifying nucleic acids encoding the biomarkers in a polymerase chain reaction. The kit may also contain nucleotides, enzymes and buffers useful in the methods of the invention as well as electrophoretic markers, such as 200bp ladder-like bands (ladders). The kit also contains detailed instructions for carrying out the methods of the invention.

The present invention also provides an algorithm-based screening assay for screening a sample of circulating fluid from a patient. Typically, input data is collected based on the level of the biomarker (or the expression level of the gene encoding the biomarker), and any increase or decrease in level is evaluated algorithmically for statistical significance, and this information is then used as output data. The present invention encompasses computer software and hardware for evaluating input data.

The assays of the invention allow integration into existing or newly developed pathology architectures or platform systems. For example, the present invention contemplates a method of allowing a user to determine a status of a subject regarding a level of placental sufficiency or placental insufficiency, the method comprising:

(a) receiving data from a user via a communication network in the form of levels or concentrations of SPINT1, SYNDECAN-1, both SPINT1 and SYNDECAN-1, or one of SPINT1 or SYNDECAN-1 with at least one other biomarker;

(b) processing the subject data via an algorithm or analytical function or analytical method or other data processing means, the algorithm or analytical function or analytical method or other data processing means providing a disease index value;

(c) determining a status of the subject based on a result of the comparison of the disease index value to a predetermined value; and

(d) an indication of the status of the subject is communicated to a user via a communication network.

Conveniently, the method generally further comprises:

(a) enabling a user to determine data using a remote end station; and

(b) data is transmitted from the terminal station to the base station via the communication network.

The base station may comprise a first processing system and a second processing system, in which case the method may comprise:

(a) transmitting the data to a first processing system;

(b) transmitting the data to a second processing system; and

The method may further comprise:

(a) transmitting the result of the algorithmic function to the first processing system; and

(b) causing the first processing system to determine a state of the subject.

In this case, the method further comprises at least one of:

(a) communicating data between the communication network and the first processing system through the first firewall; and

(b) data is transferred between the first processing system and the second processing system through the second firewall.

The second processing system may be coupled to a database adapted to store predetermined data and/or algorithms, and the method may comprise:

(a) querying a database to obtain at least selected predetermined data from the database or to access an algorithm from the database; and

(b) the selected predetermined data is compared to the subject data or a predicted probability index is generated.

Reference to an "algorithm" or "algorithmic function" as outlined above includes performing, but is not limited to, multivariate or univariate analytical functions. Other analysis functions may also or alternatively be performed. Many different architectures and platforms may be implemented in addition to those described above. It should be understood that any form of architecture suitable for implementing the present invention may be used. One technique is the use of a distributed architecture. This can improve the efficiency of the system by reducing data bandwidth costs and requirements, and ensuring that if one base station becomes congested or fails, other end stations can take over. This also allows load sharing, etc., to ensure that the system is accessible at all times.

In the above aspects, the term "data" means the level or concentration or turnover rate of the biomarker. This may be determined alone or in combination with physical, chemical or physiochemical parameters, such as data obtained from ultrasound or other physical testing procedures. The "communication network" includes the internet. When a server is used, the server is typically a client server or, more specifically, a Simple Object Application Protocol (SOAP).

The present assay may be incorporated into the present diagnostic framework as an additional pregnancy test or as a stand-alone test. For example, the assay may be associated with an ultrasound or physical measurement.

Once the information is available, the data can be used in a clinical management protocol for pregnancy. This may include a decision to deliver the baby earlier than originally planned.

Accordingly, there is provided a clinical management regimen for a pregnant mammalian subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein a decrease in the SPINT1 and/or SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 and/or SYNDECAN-1 is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, a clinical management regimen for a pregnant mammalian subject is provided, the regimen comprising determining the level of circulating SPINT1, wherein a decrease in SPINT1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, a clinical management regimen for a pregnant mammalian subject is provided, the regimen comprising determining the level of circulating SYNDECAN-1, wherein a decrease in SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in SYNDECAN-1 level is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, a clinical management regimen for a pregnant mammalian subject is provided, the regimen comprising determining the levels of circulating SPINT1 and at least one other biomarker, wherein a decrease in the SPINT1 over time compared to a control or statistically validated level and a change in the ratio of the at least one other biomarker compared to a control or statistically validated level or reflecting a decrease in the levels of the SPINT1 and the at least one other biomarker is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, there is provided a clinical management regimen for a pregnant mammalian subject, the regimen comprising determining the levels of circulating SYNDECAN-1 and at least one other biomarker, wherein a decrease in SYNDECAN-1 over time as compared to a control or statistically validated level and a change in the ratio of or reflecting a decrease in the levels of SYNDECAN-1 and at least one other biomarker as compared to a control or statistically validated level indicate placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

Also taught herein is a clinical management regimen for a neonate in a pregnant mammalian subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein an increase in the SPINT1 and/or SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting an increase in the SPINT1 and/or SYNDECAN-1 level is indicative of a neonate, wherein the fetus is monitored or undergoes early childbirth.

As noted above, although useful in veterinary medicine, in one embodiment, the mammalian subject is a pregnant human female subject. The sample tested was the circulating parent fluid.

Accordingly, there is provided a clinical management regimen for a pregnant human female subject, the regimen comprising determining the level of circulating SPINT1 and/or SYNDECAN-1, wherein a decrease in the SPINT1 and/or SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 and/or SYNDECAN-1 is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, a clinical management regimen for a pregnant human female subject is provided, the regimen comprising determining the level of circulating SPINT1, wherein a decrease in SPINT1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in the level of SPINT1 is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, a clinical management regimen for a pregnant human female subject is provided, the regimen comprising determining the level of circulating SYNDECAN-1, wherein a decrease in SYNDECAN-1 over time compared to a control or statistically validated level or a change in the rate reflecting a decrease in SYNDECAN-1 level is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, a clinical management regimen for a pregnant human female subject is provided, the regimen comprising determining the levels of circulating SPINT1 and at least one other biomarker, wherein a decrease in the SPINT1 and the at least one other biomarker over time as compared to a control or statistically validated level or a change in the ratio reflecting a decrease in the levels of the SPINT1 and the at least one other biomarker is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early labor.

In one embodiment, a clinical management regimen for a pregnant human female subject is provided, the regimen comprising determining the levels of circulating SYNDECAN-1 and at least one other biomarker, wherein a decrease in SYNDECAN-1 and at least one other biomarker over time as compared to a control or statistically validated level or a change in the ratio reflecting a decrease in the levels of SYNDECAN-1 and at least one other biomarker is indicative of placental insufficiency, and wherein the fetus is monitored or undergoes early delivery.

In one embodiment, the invention includes an assay for determining the status of a large fetus in a subject, the assay comprising determining the concentration of a biomarker selected from the group consisting of SPINT1 and SYNDECAN-1 in a circulating biological sample from the subject; subjecting the levels to an algorithm or analytical function or analytical method or other data processing means generated from a first data repository comprising levels of the same biomarker from a subject or cohort of subjects having a known status with respect to a large fetus, wherein the algorithm or analytical or data processing means provides an index of probability of an infant of which the subject has or does not have a large fetus.

As described herein, levels of SPINT1 and/or SYNDECAN-1 are indicators of fetal health, whether due to adequate or incomplete placental function, or as a marker of a large fetus. Thus, screening and determining SPINT1 and/or SYNDECAN-1 levels that are lower or higher than control is included herein to monitor or detect FGR or SGA infants (if SPINT1 and/or SYNDECAN-1 is low) or giant fetuses (if SPINT1 and/or SYNDECAN-1 is high).

As described elsewhere herein, the inventors developed and validated 4-tier risk assessment models for different low birth weight ranges based on different spinnt 1MoM cutoff levels. For example, a cycle of <0.63, SPINT1MoM (measured from about 35-37 weeks gestation) represents a high risk (grade 1); 0.63 to 1.1 represents normal risk (grade 2); 1.1 to 1.6 represent lower risk (grade 3); and >1.6 represents the lowest risk of delivering a newborn with low birth weight (grade 4). The highest ranking (rank 1) is associated with a 14.1%, 19.7%, 28.2% and 46.5% risk of a woman delivering a newborn with a birth weight <3 rd, < 5 th, < 10 th and < 20 th percentile, respectively. In contrast, the lowest ranking (rank 4) is associated with a risk of 0.0% to 6.6% of born newborns at these fetal weight percentiles. Thus, in one embodiment, a pregnant female subject is determined to be at high risk of delivering a low birth weight newborn if the subject has a circulating level of SPINT1 of less than about 0.63 (MoM). In another embodiment, a pregnant female subject is determined to be at low risk of delivering a low birth weight neonate if the subject has a circulating level of SPINT1 greater than about 1.1 (MoM).

Also provided herein is a clinical management regimen for a pregnant female subject, the regimen comprising determining a level of circulating SPINT1, wherein a circulating SPINT1 concentration of less than about 0.63MoM at about 35 to about 37 weeks of gestation is indicative of an increased risk of delivering a low birth weight infant, and wherein the fetus is monitored or undergoes early delivery.

In another embodiment, a clinical management regimen for a pregnant human subject is provided, the regimen comprising determining a level of circulating SPINT1, wherein a circulating SPINT1 concentration of less than about 0.63MoM at about 36 weeks of gestation is indicative of an increased risk of delivering a low birth weight infant, and wherein the fetus is monitored or undergoes early delivery.

As described elsewhere herein, the inventors have surprisingly found that levels of SPINT1 and SYNDECAN-1 at about 27 to 29 weeks of gestation have been significantly lower in women subsequently delivering SGA infants compared to women subsequently not delivering SGA infants. Thus, it will be appreciated that the assays, methods and protocols disclosed herein can be performed using the level of SPINT1 and/or SYNDECAN-1 measured in a sample obtained from a pregnant mammalian subject at about 27 to about 36 weeks of gestation. "about 27 weeks to about 36 weeks" includes about 27 weeks, preferably about 28 weeks, preferably about 29 weeks, preferably about 30 weeks, preferably about 31 weeks, preferably about 32 weeks, preferably about 33 weeks, preferably about 34 weeks, preferably about 35 weeks, or about 36 weeks of gestation.

Accordingly, in one embodiment, a clinical management regimen for a pregnant mammalian subject is provided. An assay, method or protocol as described herein comprises determining the level of circulating SPINT1 in a sample obtained from a subject at a time point from about 27 weeks to about 36 weeks gestation, preferably from about 27 weeks to about 29 weeks gestation, or more preferably about 28 weeks gestation.

In one embodiment, the regimen further comprises monitoring or causing early delivery of a fetus in a subject identified as having placental insufficiency and/or at risk of low birth weight.

In certain embodiments, the subject is monitored using the assays, methods or protocols of the invention to determine the presence or absence of placental insufficiency or a condition associated therewith, such as FGR or SGA or giant, to provide an indicator of whether early delivery of the fetus may be required. Monitoring is typically performed by a series of tests. In some cases, a pregnant subject is monitored as needed (e.g., on an as needed basis) using the methods described herein. Alternatively, or in addition, pregnant subjects may be monitored weekly, monthly, or at any pre-specified interval. In some cases, the pregnant subject is monitored at least once every 24 hours. In some cases, the pregnant subject is monitored at least once every 1 to 30 days. In some cases, the pregnant subject is monitored at least once every at least 1 day. In some cases, the pregnant subject is monitored at least once every up to 30 days. In some cases, pregnant subjects are monitored at least (optionally on average) once every 1 day to 5 days, 1 day to 10 days, 1 day to 15 days, 1 day to 20 days, 1 day to 25 days, 1 day to 30 days, 5 days to 10 days, 5 days to 15 days, 5 days to 20 days, 5 days to 25 days, 5 days to 30 days, 10 days to 15 days, 10 days to 20 days, 10 days to 25 days, 10 days to 30 days, 15 days to 20 days, 15 days to 25 days, 15 days to 30 days, 20 days to 25 days, 20 days to 30 days, or 25 days to 30 days. In some cases, the pregnant subject is monitored at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 29, 30, or 31 days. In some cases, the pregnant subject is monitored at least once every 1, 2, or 3 months. In some cases, the frequency of monitoring pregnant subjects via the methods described herein does not exceed 1 week, 10 days, 2 weeks, 3 weeks, or 1 month. In other words, the predictive value of some of the methods described herein may last for at least one week, at least 10 days, at least two weeks, at least three weeks, or at least one month of clinical use.

Without wishing to be bound by theory or mode of operation, it is believed that premature delivery of the fetus may be associated with greater risk of the infant suffering from complications in the short term (e.g., respiratory distress) or in the long term (e.g., IQ and attention deficit). In particular, it is known that delivery before 37 weeks of gestation carries a significantly increased risk and that a decision whether to intervene with a childbirth assisting intervention (e.g. induction of labour) may be made based on the SPINT1 and/or SYNDECAN-1 and optionally one or more other physiochemical parameters and/or risk factors. It is also known that 37-38 weeks of labour have manageable but non-zero risks and may be considered on a case-by-case basis. In contrast, delivery at about 38.5-39.5 weeks is known to be unrelated to a significant risk of fetal complications. Furthermore, clinical trials have shown that induction of labor is also not associated with increased risk of medical intervention on the mother, such as caesarean section or device assisted birth (Grobman et al NEJM 2018,379: 513-.

Thus, in particular embodiments, a pregnant subject is monitored periodically (as described with respect to the examples above) from about 35-37 weeks gestation using the assay, method or protocol of the invention, and the fetus is delivered prematurely between about 38 weeks gestation and about 39 weeks gestation, based on the results of the assay, method or protocol indicating placental insufficiency or a fetus with restricted or large fetal growth. Such intervention is thought to reduce the likelihood of stillbirth (a significant increase in the risk of stillbirth after 38 weeks gestation), fetal or maternal damage in pregnancies in which a suspected large fetus is present (pregnancy is not induced at term gestation and persists until prenatal occurs) (Boulvain et al Lancet 2015; 385(9987): 2600-. In contrast, if in these embodiments the results of the assay, method or protocol indicate that the placenta is functioning sufficiently and/or the expected body weight is within the normal range, then the fetus is not delivered prematurely and the pregnant subject is allowed to continue until late pregnancy.

As shown elsewhere herein, the inventors surprisingly found that circulating levels of SPINT1 and/or SYNDECAN-1 in pregnant female subjects indicate a risk of low birth weight, noting that low levels of SPINT1 and/or SYNDECAN-1 as early as about 27-29 weeks of gestation are at significant risk of delivering low birth weight (less gestational age) newborns. These findings can be applied to a birth weight calculator; that is, to a device or method for predicting neonatal birth weight by correlating the circulating level of SPINT1 and/or SYNDECAN-1 in a pregnant female subject with a predetermined circulating level of SPINT1 and/or SYNDECAN-1 in a female subject of known neonatal birth weight. Accordingly, the present disclosure extends to a method of predicting the birth weight of a newborn, the method comprising (i) determining the circulating level of SPINT1 and/or SYNDECAN-1 in a sample from a pregnant female subject; (ii) providing a predetermined correlation between circulating levels of SPINT1 and/or SYNDECAN-1 and neonatal birth weight in more than one pregnant female subject of the same species; and (iii) predicting the birth weight of the neonate based on the predetermined correlation of step (ii) from the circulating water level of SPINT1 and/or SYNDECAN-1 determined in step (i).

The present disclosure also extends to a device for predicting birth weight of a neonate, comprising (i) a receiver operable to receive biomarker information, wherein said biomarker information is the level of cycle of SPINT1 and/or SYNDECAN-1 in a sample from a pregnant female subject; and (ii) a birth weight determiner operable to provide an output representative of a prediction of a birth weight of a neonate using the biomarker information; wherein the predicted birth weight is based on a predetermined correlation between circulating levels of SPINT1 and/or SYNDECAN-1 and neonatal birth weight of more than one pregnant female subject of the same species.

In one embodiment, the predetermined correlation between (a) the circulating water level of SPINT1 and/or SYNDECAN-1 and (b) neonatal birth weight of more than one pregnant female subject of the same species consists of a regression coefficient (R) of about 0.020 to 0.060²) And (4) defining. In a kind of implementationIn one embodiment, the predetermined correlation is defined by a regression coefficient of about 0.026 to about 0.52. In one embodiment, the predetermined correlation is defined by a regression coefficient of about 0.52.

As described elsewhere herein, the inventors have unexpectedly discovered that circulating levels of SPINT1 in pregnant female subjects are correlated with clinical parameters of placental insufficiency such as neonatal lean body mass (R)²＝0.064；p<0.0001) and placenta weight (R)²＝0.087；p<0.0001). Accordingly, the present disclosure extends to an assay for determining placental weight and placental surface area in a pregnant female mammalian subject, the method comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease over time or a change in ratio relative to a control or a change over time is indicative of a lower placental weight and a lower placental surface area. Also embodied herein is a clinical management regimen for a pregnant mammalian subject, the regimen comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in the concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease over time or a change in the ratio relative to a control or a change over time indicates a lower placental weight and lower placental surface area, and wherein the fetus is monitored or undergoes early delivery.

The present disclosure also extends to an assay for determining lean body mass of a neonate, the method comprising determining circulating levels of SPINT1 and/or SYNDECAN-1 in a pregnant female mammalian subject, wherein a decrease in concentration of SPINT1 and/or SYNDECAN-1 relative to a control or a decrease over time or a change in ratio relative to a control or a change over time indicates that the neonate has lower lean body mass. Also embodied herein is a clinical management regimen for a pregnant mammalian subject, the regimen comprising determining maternal circulation levels of SPINT1 and/or SYNDECAN-1, wherein a decrease in concentration or a decrease over time or a change in ratio or a change over time of the concentration of SPINT1 and/or SYNDECAN-1 relative to a control indicates that the neonate has a lower lean body mass, and wherein the fetus is monitored or undergoes early delivery, and/or the subject is exposed to a treatment to increase lean body mass of the neonate.

Examples

The aspects disclosed herein are further described with reference to the following non-limiting examples.

Example 1

Placenta-specific proteins

Fetal Longitudinal Growth Assessment (FLAG) studies were designed to examine the predictive ability of measured mRNA encoding placenta-specific genes and their proteins in maternal blood at 28 and 36 weeks of gestation for term FGR. Longitudinal samples were collected from 2000 pregnant women; 10.5% subsequently delivered SGA (< 10 th percentile) infants. A 1:1 case control set (i.e., 105 SGA cases and 105 matched controls) was systematically selected for 28 of the 51 surface proteins from the first 1000 FLAG samples over 18 months using ELISA. SPINT1 was identified as a promising new candidate marker for placental insufficiency.

SPINT1 is a protease inhibitor that regulates Cell surface and extracellular serine proteases involved in tissue remodeling (Tanaka et al (2005) Mol Cell Biol 25: 5687-5698). Sprit 1 was originally identified as an inhibitor of hepatocyte growth factor activator. It is also known as HAI-1. The importance of SPINT1 in placental development is highlighted by the fact that: due to failed placental development and function, SPINT1 knockout mice have severe growth limitations and embryonic lethality (Tanaka et al (2005) supra). The SPINT1 knockout mouse has an impaired labyrinthine layer formation, which is critical in maternal/fetal exchange. Until now, the potential role that altered SPINT1 might have in human FGR has not been considered in the art. Another placenta-specific protein is SYNDECAN-1. The present invention identifies SPINT1 and SYNDECAN-1 as novel markers of placental insufficiency.

Example 2

SPINT1 and SYNDECAN-1 as markers for FGR

SPINT1 performed better than placental growth factor (PlGF) as a marker for FGR. To date, PlGF has been considered as the strongest biomarker for placental insufficiency. PlGF is highly expressed by the placenta and decreased circulating levels are associated with FGR (Benton et al, (2012) Am J Obstet Gynecol 206(163): e 161-167; Griffin et al, (2015) Ultrasound in obstercs and genetics 46: 182-.

Measuring PlGF alone does not yield a diagnostic test that is sufficiently effective to integrate into clinical care. Measuring PlGF at 30-34 weeks plus assessing maternal risk factors has only a sensitivity of 58% for identifying SGA infants delivered within 5 weeks and only a sensitivity of 34% (specificity of 90%) for SGA infants delivered over 5 weeks [ Bakalis et al (2015) Ultrasound in obstercs and gynecomology 46: 2089-. PlGF levels are also significantly lower in women with infants with birth weights < 5 th percentile when measured at 35-37 weeks gestation, but PlGF levels are evaluated for a single maternal risk factor for a surprisingly small predictive power for detecting increases in the predictive power of SGA (Fadigas et al (2015) Ultrasound in obstetrics and gynecomogy 46: 191-197). Refer to fig. 1A to 1P. Next, results from the case control cohort were validated in a sample set consisting of 1004 samples collected at 36 weeks gestation. 920 controls and 84 cases where the mother subsequently delivered SGA infants. Circulating SPINT1 was significantly reduced in women who subsequently delivered SGA infants, with an AUC of 0.74 (FIGS. 1I, 1J). Notably, the AUC of the SPINT1 was not reduced — it was 0.74 compared to 0.75 for the case/control set. Similarly, SYNDECAN-1 remained significantly reduced in women delivered SGA infants, with an AUC of 0.65 (fig. 1K, 1L).

Next, the aim was to assess whether these proteins are likely to decrease in maternal circulation in the early stages of pregnancy in those destined to have SGA fetuses. To this end, a case control cohort was selected and we measured circulating SPINT1(n 130 controls, n 104 SGAs) and SYNDECAN-1(n 100 controls, n 84 SGAs) at 28 weeks gestation. At 28 weeks gestation, SPINT1 in women who subsequently delivered SGA infants had been significantly reduced, with an AUC of 0.69 (fig. 1M, 1N). Similarly, SYNDECAN-1 was significantly reduced in those destined to deliver SGA fetuses at 28 weeks gestation, with an AUC of 0.69 (fig. 1O, 1P). This is a particularly interesting finding considering that women with reduced plasma SPINT1 and SYNDECAN-1 may not deliver their SGA infants until after 10-12 weeks. Notably, PlGF levels at 28 weeks gestation did not change significantly. Data are expressed as mean +/-SEM-each symbol represents individual patient,. + -. p < 0.0001.

When the circulating SPINT1 concentration was measured in 1000 women, it was found to be strongly correlated throughout the successive birth weight percentiles (2A, 2B), indicating a close continuous relationship between circulating SPINT1 levels and fetal size.

The body fat mass and lean body mass at birth of a subset of infants was measured using a peapod machine, and the body fat percentage was also estimated by measuring the subcutaneous fat at the level of the midtriceps (mid-triceps) and the subscapular region of the infant with calipers. SPINT1 was found to be associated with neonatal lean body mass (2C), but not with the percent skin fold body fat (2D).

Taken together, these data provide convincing evidence that circulating SPINT1 and SYNDECAN-1 are clinically useful biomarkers of fetal growth.

Example 3

Reduction of SPINT1 in FGR placenta and SPINT1 by hypoxia

This example determines whether SPINT1 in FGR placenta is reduced and whether this is the cause of a reduced circulating water level. Fig. 3A-3P are graphical representations showing an observational study to determine whether placental SPINT1 has changed in human SGA cases. To this end, a cohort of placentas was selected from controls, preeclampsia or SGA pregnancies. The SPINT1mRNA was measured using quantitative RT-PCR and showed significant reductions in SPINT1mRNA in both preeclampsia and SGA cohorts compared to controls in patients who delivered their infants <34 weeks gestation. No changes in SPINT1mRNA expression were detected in the placenta collected from patients delivered at >34 weeks gestation. The SPINT1 protein expression was assessed using western blot and commercially available antibodies. In samples collected both at <34 weeks gestation and >34 weeks gestation, the SPINT1 protein in the placenta of women from SGA-delivered infants was significantly reduced. Data are presented as mean +/-SEM-each symbol represents individual patient or mouse, # p <0.05, # p <0.01, # p <0.001, # p < 0.0001.

Given that placental insufficiency is characterized by chronic placental hypoxia, it was next assessed whether hypoxia would reduce placental SPINT1 expression. Exposing primary human trophoblasts (i.e., placental cells isolated from freshly delivered placenta) to 1% (hypoxic) or 8% (normoxic) oxygen (O)₂)。

The next step was to assess whether exposure of primary placental cells (cytotrophoblast cells isolated from full-term human placenta) to hypoxic conditions (1% v/v oxygen for hypoxia, and 8% v/v oxygen for normoxia) would alter SPINT1mRNA and protein expression and protein secretion. mRNA expression was initially assessed by qRT-PCR, and we found that exposure of primary placental cells to hypoxia resulted in significantly reduced SPINT1mRNA expression (3E), and a similar finding was observed for cellular proteins measured by western blot (3F). Next, secretion of SPINT1 into the medium bathing the placental cells was measured using the same ELISA as for plasma SPINT1 in blood, and we found that when the placental cells were made hypoxic, the level of SPINT1 in the medium was also significantly reduced (3G). Thus, strong evidence was obtained demonstrating that placental hypoxia reduces SPINT1 transcription and protein production.

The SPINT1 was evaluated in a FGR mouse model caused by maternal hypoxia. In this model, pregnant mice were exposed to hypoxia (10% inhaled O) at E14-19₂) Or normoxia (21% inhaled O)₂) [ Higgins et al (2016) The Journal of physiology 594:1341-1356]Then slaughtered (cubed) at E19.5 (note that the term of the mice is about day 20). Exposure of pregnant females to hypoxia significantly impairs vagal zone formation (equivalent to the placental interface in the human placenta), reduces the surface area and density of fetal capillaries, and impairs matrix exchange and blood flow to the growing fetal-placental unit. First, it was shown that there were hypoxia-induced growth-restricted fetuses in the placenta where the SPINT1mRNA was measured, but there was no change in placenta weight (3H, 3I). It was subsequently shown that in mouse placenta exposed to hypoxia, there was indeed a significant reduction in placental SPINT1mRNA expression (3J). Placental sprint 1 protein expression was then measured. We again demonstrated that this hypoxia reduced fetal weight, but not placental weight (3K, 3L). In this group of animal studies, we demonstrated that the placental SPINT1 protein was also significantly reduced under hypoxia (3M). Thus, strong evidence was obtained indicating that SPINT1 is reduced in the placenta of SGA infants, and that a reduction in SPINT1 may be associated with placental hypoxia.

Thus, these data indicate that hypoxia reduces placental SPINT1 expression and that SPINT1 is significantly reduced in the placenta of growth-restricted fetuses. Thus, the reduced circulating SPINT1 concentrations observed in our FLAG cohort are likely of placental origin.

The inventors next set out to evaluate whether silencing of the SPINT1 using siRNA knockdown in the HTR8 placental cell line would affect cell proliferation assessed in real time using the xcelligene system. Indeed, when SPINT1 was silenced in HTR8 cells, impaired proliferation was found (3N).

The inventors tried to determine if reduced proliferation could be the result of increased apoptosis, silencing the SPINT1 in HTR8 cells, then collecting the protein and measuring the apoptosis markers BAX, BCL2 and cleaved caspase 3(3O) by western blot. Although the SPINT1 protein expression was found to be significantly reduced as expected (the upper panel shows the lack of bands under sipint), there was no significant change in expression of apoptosis markers, suggesting that reduced proliferation when SPINT1 was silenced was not a consequence of increased apoptosis. Beta-actin was used as loading control.

The inventors also sought to determine whether enhancing SPINT1 would alter HTR8 placental cell line proliferation. To this end, a commercially available SPINT1 mimetic (Glixx laboratories) designated SRI31215 was applied to HTR8 cells and proliferation was monitored using the xcelligene system. SRI31215 at 5uM or 10uM was found to enhance HTR8 proliferation.

Example 4

SPINT1 critical to normal placental function

The purpose of this embodiment is: characterizing expression of a proteolytic enzyme (matriptase) in the FGR placenta and assessing the effect of placental SPINT1 reduction on proteolytic enzyme activity in isolated trophoblast cells; assessing the effect of reduced placental SPINT1 on normal human placental cell function, including trophoblast integration (recruitment), invasion and proliferation; and a conditional placental SPINT1 knock-out mouse model was used to characterize the precise timing of the SPINT1 dependence of normal placental development.

General experimental methods: normal and FGR placental samples were collected from women delivered by caesarean section (to avoid confounding effects of delivery (labour)). Early pregnancy samples were obtained from cases where pregnancy was selectively terminated. Two placental primary tissue types were used: 1) human placental explant (5 mm)³Placental mass) and 2) primary isolated trophoblast cells from full or early gestation placenta (Kaitu' u-Lino et al (2014) Pregnancy hypertension 4: 287-295). Equal numbers of placentas were obtained from both male and female pregnancies. Details of methods for trophoblast isolation, placental explant culture, RNA extraction, qPCR, or Western blotting are described in Kaitu' u-Lino et al (2014), supra; brownfoot et al, (2015) Am J Obstet Gynecol; brownfoot et al (2015) Hypertension 66: 687-697; kaitu' u-Lino et al (2)012) The American journal of Pathology 180: 888-894; tong et al (2015) Hypertension 66: 1073-1081; onda et al, (2017) Hypertension 69: 457-468; kaitu' u-Lino et al, (2017) Hypertension 70: 1014-1024.

Statistics (for the entire document): comparing normal distribution data using parametric tests; non-normal distribution data were compared using a non-parametric test. The primary trophoblast experiment was repeated 4-5 times, and the placental explant study was repeated 5-7 times (because of the increased variability). Biological replicates were performed on samples from different patients, and each experiment was performed in triplicate. Statistical analysis was performed on the median (or mean) from biological replicates. For animal studies, measurements were repeated using a linear mixed model, which takes into account that each fetus was a duplicate measurement in a litter.

Characterization of proteolytic enzyme expression in FGR placenta and evaluation of the effect of placental SPINT1 reduction on proteolytic enzyme activity in isolated trophoblast cells. As protease inhibitors, SPINT1 targets 3 known proteases-one of which is a proteolytic enzyme. The spinnt 1 knockout in mice results in failed placental development, embryonic lethality, and elevated levels of proteolytic enzymes. Simultaneous knock-out of SPINT1 and proteolytic enzymes rescued placental development and embryonic lethality, demonstrating that increased proteolytic enzyme activity contributes to placental deficiency and embryonic lethality (Szabo et al, (2007) Oncogene 26: 1546-1556; Szabo et al, (2014) PLoS genetics 10: e 1004470). mRNA, protein expression and localization of proteolytic enzyme in FGR placenta were characterized (same cohort used in fig. 2). The effect of silencing spinnt 1 on the activity of proteolytic enzymes in human placental cells was evaluated. SPINT1 in isolated primary trophoblasts was silenced using siRNA and the proteolytic activity was measured using a commercially available assay to determine if the activity was increased as observed in mouse placenta. Term trophoblasts and early pregnancy trophoblasts were evaluated. A second model of reduced spinnt 1 was induced by placental hypoxia. Exposure of isolated primary trophoblasts to hypoxia (1% oxygen) reduced SPINT1 (fig. 3). Primary trophoblast and placental explants were exposed to hypoxia (1% oxygen) or normoxia (8% oxygen) to reduce SPINT1 expression and to measure the effect on proteolytic activity.

This provides the first evidence that reduced SPINT1 results in excessive proteolytic activity in primary human placental cells.

The effect of reduced placental SPINT1 expression on placental cell invasion, migration, and trophoblast integration was evaluated. Placental insufficiency and FGR are associated with poor placental growth, including reduced trophoblast invasion, migration and proliferation. Furthermore, evidence from mouse models (Tanaka et al (2005) supra; Szabo et al (2007) supra) suggests a crucial role for SPINT1 in the establishment of the maternal/fetal interface, i.e., the maze area. The effect of spinnt 1 knockdown on trophoblast invasion, migration and proliferation was evaluated. Evidence is provided to demonstrate that silencing SPINT1 in trophoblast cell line HTR8 cells results in reduced proliferation. The effect of SPINT1 knockdown (using siRNA) on HTR8 and early pregnancy trophoblast migration, invasion and proliferation was evaluated using xcelligene. Trophoblast syncytia are first evaluated in isolated term trophoblasts. These cells spontaneously underwent trophoblast integration in culture (Kaitu' u-Lino et al (2014) supra). The silencing of SPINT1 using siRNA and the effect on trophoblast integration over time were measured by measuring markers of the process (including human chorionic gonadotropin; via ELISA) and the loss of E-cadherin (PCR, Western blot and immunofluorescence). It was also determined whether placental sprit 1 decreased increased apoptosis (caspase 3 and 9, BCL2 and Bax expression, via western blot).

This demonstrates that loss of spinnt 1 expression in human placental cells results in impaired placental cell function and development.

A conditional placental SPINT1 knockout mouse model was used to characterize SPINT1 in normal mouse placental development. The present inventors could promote the specific knock-down or overexpression of genes in mouse placenta by lentivirus transduction of genes (or shrnas) into cells that continue to form the placenta (trophectoderm) [ Onda et al (2017) supra ]. The transduced blastocysts were then transferred to recipient dams (Onda et al, (2017 supra; Kumasawa et al, (2010) Proc Natl Acad Sci USA) allowing the formation of a genetically modified placenta with the mother and young intact. This model is intended to silence the SPINT1 only in the placenta.

Data from Tanaka et al (2005) supra and Szabo et al (2007) supra demonstrate that knockout of SPINT1 in mice results in failed placental development and growth limitation at day (E)9.5 of the embryo and lethality between days E10.5-12.5. Conditional lentiviruses are used that can knock down the expression of placental SPINT1 at specific predetermined intervals throughout pregnancy. This is achieved by including an inducible tetracycline promoter in the lentiviral plasmid backbone, which is induced only when the mother receives doxycycline (Fan et al (2012) Endocrinology 153: 5637-5644). This technique is demonstrated using inducible promoters. A fluorescent tagged doxycycline-responsive transactivator protein with high sensitivity to doxycycline was used (Fan et al (2012) supra; Moutier et al (2003) Transgenic research 12: 369-. In mice, the maze area develops from about E8.5-9, when villi begin to extend and branch (Anson-Cartwright et al (2000) Nat Genet 25: 311-314). In view of the lack of maze regions in the SPINT1 knockout mice (Tanaka et al (2005) supra), doxycycline was initially taken into drinking water at E6.5, E8.5, E9.5, E10.5, E11.5, and E12.5; the uptake of doxycycline-containing water activates lentiviruses, resulting in silencing of the SPINT1 expression at these specific pregnancies. Two control groups were used: 1) mice with blastocysts containing empty lentivirus transferred to serve as controls for any doxycycline effect; and 2) mice transfected with SPINT1 shRNA but not receiving doxycycline. Pregnant dams were slaughtered 4 days after the introduction of doxycycline or vehicle control to assess: 1) placental and circulating SPINT1mrna (pcr) and protein expression (western blot) to confirm knockdown; 2) fluorescent localization to confirm trophoblast transfection; 3) placenta size and morphology; and 4) litter size, fetal growth (including fetal weight, crown-hip length, limbs, and liver and brain weight to assess growth-limiting symmetry and brain protection effects). Placental parameters, including labyrinth development markers (e-cadherin and β -catenin) were measured, and trophoblast density, maternal blood space (blood space), and fetal blood vessels were evaluated.

This determined that accurate pregnancy windows of SPINT1 were required for placental development and established a new fetal growth restricted mouse model. Together, these data demonstrate that reduced placental SPINT1 increases the expression and activity of protease proteolytic enzymes in human cells, which in turn impairs placental development. In addition, a conditional SPINT1 knockdown mouse model was developed that serves as a novel FGR mouse model that can be used to test potential treatments.

Example 5

SPINT1 as a potential diagnostic marker for placental insufficiency

SPINT1 is a novel marker for FGR. Improving FGR detection in all stages of pregnancy is a key first step in improving perinatal outcomes, but there is currently a lack of reliable screening strategies. As outlined previously, SPINT1 has great potential as a clinically useful diagnostic biomarker, significantly decreasing in the FLAG cohort as early as 12 weeks before delivery of full-term SGA infants. While PlGF is one of the most widely studied markers of placental insufficiency, data from FLAG suggests that it does not perform as well as SPINT 1. In recent years, the ratio of PlGF and the anti-angiogenic molecule sFlt-1 has been evaluated to determine the potential of its biomarkers, and it has been reported that this ratio is associated with an increased risk of stillbirth (chaiwoapagnga et al (2013) Obstet Gynecol 208(287): e281-287, e 215). PlGF and sFlt-1 were measured using 36-week samples and the sensitivity of the test (at a fixed specificity of 90%) as biomarker was determined. It was found that although the PlGF or sFlt1/PlGF ratio showed limited (modest) prediction of FGR (sensitivity of 29% and 27%, respectively), SPINT1 alone already had a sensitivity of 40%. Interestingly, the addition of SPINT1 to the sFlt1/PlGF ratio significantly improved the detection of infants destined to be born as SGA to 48%: more than twice the number detected with current conventional care (tape measure and selective ultrasound) and in fact approaches the detection rate of general purpose ultrasound (Paiva et al (2011) J Clin Endocrinol Metab 96: E1807-1815; Moutier et al (2003) supra). Thus, it was proposed that SPINT1 in combination with sFlt1/PlGF represents a clinically useful blood test.

FLAG data indicate that SPINT1+/-sFlt1/PlGF is expected to be a useful biomarker for full-term SGA. This finding is worth validating in an independent cohort, but it is also useful to determine whether this biomarker approach has clinical utility in improving the detection of preterm FGR. This is important because these pregnancies benefit from intensive monitoring and this is a candidate for trials for future therapeutic intervention. Thus, a queue is used that provides an opportunity to determine whether the SPINT1 at 28 weeks is a useful predictor of SGA, including those with severe early-onset FGR. Measuring PIGF +/-sFlt1 also allows us to assess whether it can improve the predicted performance of these proposed biomarkers. It is believed that the SPINT1 was lower at 28 weeks among those in the cohort who were destined to deliver SGA fetuses.

Example 6

SYNDECAN-1

See fig. 4A-4C. As observed for the print 1, the circulating maternal SYNDECAN-1 (expressed as median of the mean) measured at 36 weeks gestation appeared to be positively correlated with the birth percentile. Fetal parameters at birth were also evaluated and it was found that circulating maternal SYNDECAN-1 measured at 36 weeks gestation was significantly correlated with both neonatal lean body mass and percent skinfold body fat.

Fig. 5A-5E are graphical representations showing an observation study to determine whether placenta SYNDECAN-1 changes in human SGA cases. To this end, a cohort of placentas (same cohort used to measure SPINT 1) was selected from control, Preeclampsia (PE) or SGA pregnancies. SYNDECAN-1mRNA was measured using quantitative RT-PCR and showed a significant increase in placental SYNDECAN-1mRNA in both the PE and SGA cohorts compared to controls in patients who delivered their infants <34 weeks gestation. Protein expression was then measured. Commercial antibodies used for western blotting yielded 3 different bands that could correspond to different isoforms of SYNDECAN-1. Densitometric analysis of these bands showed that the 85kDa and 80kDa bands were unchanged, while the 33kDa band was significantly reduced in the SGA placenta. SYNDECAN-1mRNA expression was subsequently measured in the placenta delivered at >34 weeks gestation and no significant changes in expression were found. Data are presented as mean values +/-SEM-each symbol represents individual patient p <0.05, p <0.01, p <0.001, p < 0.0001.

In view of the correlation between SGA and placental hypoxia, fig. 6A-6F are graphical representations of the effect of placental hypoxia on syndecanon mRNA and protein expression using the same sample from fig. 3 for the determination of SPINT 1. qRT-PCR showed that hypoxia induced a significant decrease in SYNDECAN 1mRNA expression (a). Although western blot showed no significant change in total protein expression (B), SYNDECAN-1 protein secretion from primary placental cells exposed to hypoxia was significantly reduced (C).

Murine SYNDECAN-1 expression in the placenta obtained from pregnant mice exposed to hypoxia and correlation with the small fetus was also measured. In those same samples where we found reduced expression of murine SPINT1, we found no significant changes in syndecano-1 mRNA (6D) or protein expression (6E).

When SYNDECAN-1 in placental cell line HTR8s was silenced (using siRNA), there was reduced cell proliferation (6F; measured using the xCELLigence system).

Example 7

Giant

It is proposed herein that elevated levels of SPINT1 and/or SYNDECAN-1 are indicative of a large fetus. The levels of one or both biomarkers are detected and measured as described herein, either alone or in combination with the levels of other biomarkers and/or physiochemical data (such as data from ultrasound) and results related to fetal or postpartum weight. Overweight infants, including those weighing more than 4kg at birth, are considered to be large infants. The ability to detect potentially large infants in utero may aid in clinical pregnancy management.

Example 8

SPINT1 is a diagnostic marker for placental insufficiency

The study investigated prospective collections from a third-degree referral hospital of melbourne australia from 28 weeks of gestation (27)⁺⁰–29⁺⁰) And 36 weeks (35)⁺⁰–37⁺⁰Day) of pregnancy in a pregnant womanCorrelation between INT1 and various clinical parameters of placental insufficiency. The study was approved by the institutional Health Research Ethics Committee (Mercy Health Research Ethics Committee) (Ethics approval No. R14/12) and written informed consent was obtained from all participants.

The cohort was split roughly in half to discover and subsequently validate biomarkers, including SPNT 1. Samples from the first 997 consecutively enrolled participants constituted queue 1 (table 2) and samples from the second 999 consecutively enrolled participants constituted queue 2 (table 3).

Table 2: maternal characteristics and pregnancy outcomes of cohort 1. Data is expressed as mean (standard deviation) if it is normal distribution data, median [ 25 th-75 th percentile ] if it is not normal distribution data, and data is expressed as number (%) if it is classified data. Less gestational age is defined as birth weight < 10 th percentile:

table 3: maternal characteristics and pregnancy outcomes of cohort 2. Data are expressed as mean (standard deviation) if it is normal distribution data, median [ 25 th-75 th percentile ] if it is not normal distribution data, and number (%) if it is classification data. Less gestational age is defined as birth weight < 10 th percentile:

347 infertile women in the FLAG study were also enrolled for a more thorough study (FLAG B cohort). They underwent ultrasonic assessment at 36 weeks gestation to measure blood flow resistance of the uterus, umbilical cord and middle cerebral artery of the fetus. Neonatal body composition (lean body mass and fat mass) was measured, where possible, within 4 days of birth by an air displacement plethysmography study using a PEAPOD device.

Women were eligible for screening and invited to participate in their oral glucose tolerance test, which is usually provided at about 28 weeks gestation, to test for gestational diabetes. English-speaking women over 18 years of age, with fetal morphological examinations of single and normal mid-term pregnancies, are eligible for participation. At the time of blood sampling, samples from women suspected of having an SGA fetus were not excluded. Participants donated blood samples at weeks 27+0 to 29+0 and/or 35+0 to 37+0 of pregnancy (endpoints included). The whole blood was collected in a 10ml ethylenediaminetetraacetic acid tube. The plasma was stored at-80 ℃ until the time of sample analysis.

Maternal characteristics and pregnancy outcomes were obtained by a clinician blinded to any protein level after reviewing the medical history, study results and hospital database entries of each participant. Assigning a customized percentile for infant birth weights using GROW software 1(www.gestation.net), the GROW software 1 generating a "term optimal weight" based on an optimized fetal weight criterion that is adjusted according to: height, weight and number of births of the mother; the gender of the infant; and the exact gestational age. The coefficients of the australian GROW dataset are known from the local dataset; the multiple regression model has a constant, which is weighted up or down for each adjusted variable. SGA is defined as the customized birth weight < 10 th percentile. Circulating protein levels in SGA cases were compared to controls.

Some of the infertile participants also participated in the ultrasound-based FLAG study group (arm), referred to herein as FLAG B. For this purpose, 36 (35) were carried out on 347 women⁺⁰-37⁺⁰) Peripheral ultrasound assessment, in which transabdominal color and pulse wave doppler were used to measure the mean maternal uterine artery Pulsatility Index (PI) and umbilical artery PI. Measurements were taken during periods of fetal apnea and inactivity, where the angle of insonation was close to zero. The umbilical artery PI is measured in the free loop of the umbilical cord (free loop of the umbilical cord) away from the umbilical cord insertion site (cord insertion site). For the maternal uterine artery, a probe was placed in each iliac fossa and waves were recorded within 1cm of the uterine artery passing through the external iliac arteryAnd (5) forming a shape 2. PI values were measured in triplicate and the average was calculated. Average uterine artery PI values were obtained for both the right and left vessels and averaged to provide an overall average PI. For each PI value, the percentile of pregnancy dependence (if normally distributed), or median fold (MoM) was determined. The treatment clinician was blinded to the uterine artery PI results.

Using data from cohort 2 (validation runs of markers found in cohort 1), diagnostic performance of potential markers was measured, alone or in combination, to predict a birth weight percentile < 20 th, < 10 th, < 5 th and <3 rd; and neonates with birth weights < 5 th percentile at birth but also requiring nursing admission (nursery administration). Specificity was set at about 90%, which corresponds to a screening positive rate of 10%.

Ultrasound was performed in a subset of 347 infertile women (distributed across cohort 1 and cohort 2) at 36 weeks gestation, followed by air displacement plethysmography (PEAPOD) of postnatal neonates (FLAG B). The SPINT1 was negatively correlated with the uterine arteries measured at 36 weeks gestation (fig. 7a), but not with the umbilical artery doppler velocity (fig. 8a), and positively correlated with neonatal lean body mass (fig. 7b), but not with fat mass (fig. 8b, fig. 8 c). SPINT1 was also strongly associated with placental weight (fig. 2 c). In contrast, the correlation between PIGF and these indicators of placental insufficiency was either not significant or more limited (fig. 9). We also found that plasma SPINT1 concentrations decreased gradually between the control (> 10 th percentile) and the decreasing birth weight percentile (fig. 7 d). Plasma SPINT1 concentrations at 36 weeks were continuously correlated across all birth weight percentiles (fig. 7 e).

These data indicate that circulating SPINT1 concentrations correlate with several clinical parameters associated with placental insufficiency in high risk cohorts from different countries.

Example 9

SYNDECAN-1 and SPINT1 were verified as diagnostic markers for placental insufficiency

The data presented in this study illustrate the correlation between SPINT1 or SYNDECAN-1 and various clinical parameters of placental insufficiency in a larger sample cohort. These data include some of the data described above, which is extended to an entire queue including up to 2040 women.

When SPINT1 was validated in the entire cohort of n-1996 samples at 36 weeks (1785 controls, 211 SGA cases), the data demonstrated a significant decrease in concentration of SPINT1 in women who subsequently delivered SGA infants (fig. 10A), with an AUC of 0.688 (fig. 10B). When examining the entire queue in n-1996, a strong correlation with the birth percentile was confirmed (fig. 10C-fig. 10D).

Similarly, when SYNDECAN-1 was validated throughout the cohort, the concentration of SYNDECAN-1 was significantly reduced in women who subsequently delivered SGA infants (fig. 10E), with an AUC of 0.61 (fig. 10F). The data also shows the correlation to the birth percentile across the entire queue (fig. 10G-10H).

The data shown in fig. 11A-11C validated the SPINT1 change at 28 weeks in the entire cohort of n-2040 (n-1827 controls, n-213 cases). The data demonstrate a significant reduction in circulating SPINT1 at 28 weeks in women delivering SGA infants (fig. 11A), with an AUC of 0.60 (fig. 11B). At 28 weeks gestation, SPINT1 was consistently associated with the birth weight percentile (fig. 11C).

The data shown in fig. 12A-12D demonstrate the change in plasma SPINT1 at 36 weeks in the larger cohort and show a correlation with markers of placental insufficiency. Plasma SPINT1 concentrations at 36 weeks gestation were correlated with Uterine Artery (UA) doppler flow resistance (fig. 12A, n ═ 325), neonatal lean body mass (fig. 12B, n ═ 281), and placental mass (fig. 12C, n ═ 378). The plasma concentration of SPINT1 was gradually reduced in women whose infants were subsequently born with birth weights below the 10 th percentile (fig. 12D).

The data shown in fig. 13A to 13D demonstrate the changes in plasma SPINT1 and SYNDECAN-1 in separate cohorts of samples collected from the day of female delivery of their infants. SPINT1 was significantly reduced in women carrying SGA infants (infants less than the 10 th percentile; FIG. 13A, FIG. 13B, n-47 SGA and n-509 control). Similarly, SYNDECAN-1 was also significantly reduced in the same women carrying SGA infants (fig. 13C, 13D).

Example 10

Developing and validating diagnostic tests to predict low birth weight-4 grade risk assessment models

Based on different spinnt 1MoM cutoff levels, 4-level risk models for different low birth weight ranges were developed (cohort 2) and then validated (cohort 1). These cutoff levels are arbitrarily chosen when testing is developed in queue 2. The investigator had prospective consent to set these thresholds, which were then applied to cohort 1 for validation. For statistical analysis, data were summarized as mean (SD), median [ 25-75 percentile ], median (min, max), and number (%) based on the distribution. Hypothesis testing between SGA states uses the Mann-Whitney rank sum test for continuous data, and Fisher exact test for categorical data. Predictive performance, expressed as point estimates and 95% confidence intervals based on Wilson, was evaluated using the area under the receiver operating characteristic curve (ROC area), sensitivity at 90% specificity, and Positive Prevalence (PPV) and Negative Prevalence (NPV) of the data. The significance level was set to 0.05 and was not adjusted for multiple comparisons. The statistical Software used was Stata v15(Stata Corp.2017.Stata statistical Software: issue 15.College State, TX: Stata Corp LLC) and the diagt program (Summary statistics for diagnostic tests. P.T.seed and A.Tobias. reprinted in Stata Technical Bulletin Reprints, vol.10, pp.90-93. from http:// fmwww.bc.edu/RePEc/bocode/d, last visit 2018, 11 months and 1 days) or Graphpad Prism 6(Graphpad Software LA, Jolla, CA).

Circulating SPINT1 concentrations <0.63MoM at 36 weeks gestation in cohort 2, a group with an increased risk of delivering low birth weight infants was identified (table 1). Notably, 46.5% of the positive screenings had a birth weight percentile < 20. We validated the diagnostic test performance of this SPINT1 cutoff in cohort 1 from the FLAG study (table 4).

Table 4: SPINT1MoM <0.63 diagnostic performance in detecting various degrees of low birth weight neonates at birth. Queue 2(n 999) is used to set the threshold of SPINT1MoM to <0.63, and queue 1(n 997) is used to verify the test performance of the set threshold.

Using queue 2, a 4-level risk model was developed based on different spinnt 1MoM concentrations (table 5).

Table 5: development and validation of a 4-tier risk model for neonatal delivery with different low birth weight cut-offs. The risk model was developed by selecting different thresholds for circulating levels of SPINT1MoM concentration measured in pregnant women at weeks 35+0-37+0 of gestation in cohort 2. The same set threshold is then applied to queue 1.

Those at the highest risk level (7.1% of cohort 2, with the lowest SPINT1 MoM) had a risk of delivering newborns with birth weights <3 rd, < 5 th, < 10 th and < 20 th percentiles of 14.1%, 19.7%, 28.2% and 46.5%, respectively. In contrast, those at the lowest risk level (9.1% of cohort 1) had a risk of delivering <3 rd, < 5 th, < 10 th and < 20 th percentile of newborns of 0.0%, 1.1% and 6.6%, respectively. The model was validated in cohort 1 with very similar diagnostic performance (table 7).

If the circulating SPINT1 concentration is within grade 1 (highest risk), the rate of delivery of infants in these birth weight percentiles is increased by a factor of 2-5 compared to the baseline population prevalence in the cohort. They were similar to the background population prevalence of grade 2, with a population prevalence of about half in grade 3 and very low in grade 4 (table 7).

These data demonstrate the development and validation of a 4-tier risk model that identifies a high-risk or low-risk cohort of newborns at low birth weight percentile delivery, where it is imperative to also identify unborn fetuses that are low in weight and at increased risk of stillbirth. This 4-tier risk model enables slightly less than half of the population under test to be assigned risk levels (tier 1, tier 3 and tier 4) that differ from the baseline prevalence. Such a 4-tier risk model may be useful in clinics, where those in tier 1 may be provided with a planned due delivery, and those in tiers 3-4 may be reassured.

Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure encompasses all such variations and modifications. The disclosure also contemplates all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features or compositions or compounds.

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