WO2021123830A1

WO2021123830A1 - Method of determining risk of fetal size abnormality

Info

Publication number: WO2021123830A1
Application number: PCT/GB2020/053312
Authority: WO
Inventors: Gordon Campbell Sinclair SMITH; David Stephen Charnock-Jones; Ulla SOVIO
Original assignee: Cambridge Enterprise Limited
Priority date: 2019-12-20
Filing date: 2020-12-18
Publication date: 2021-06-24
Also published as: GB201919092D0

Abstract

The present invention provides metabolites and methods useful for screening for risk of fetal size abnormality in a subject.

Description

Method of determining risk of fetal size abnormality

FIELD OF THE INVENTION

The invention relates to metabolites and methods for screening for risk of fetal size abnormality (FSA) in a subject. The invention relates to metabolites and methods for screening for risk of fetal growth restriction (FGR) in a subject, as well as metabolites and methods for screening for risk of large for gestational age (LGA) in a subject. The invention also relates to computer programs for performing said methods, and to methods of treating a subject identified as being at risk.

BACKGROUND OF THE INVENTION

Estimation of fetal weight is routinely performed during pregnancy to help determine whether the fetal weight is appropriate for gestational age. Fetuses that are large for gestational age (typically in the 90^th percentile for body weight) or fetuses that are small for gestational age (typically in the 10^th percentile for body weight) are at increased risk of complications during pregnancy and childbirth, and it is critical that abnormal fetal size is identified as early as possible. Interventions for FSA (such as caesarean section and early induction) can significantly improve outcomes, and so an accurate and reliable diagnostic tool can provide considerable clinical advantages. Current methods for identifying FSA (such as ultrasound) are frequently associated with undesirably high false negative or false positive rates, which can result in unnecessary and potentially harmful intervention, or the presence of FSA being overlooked altogether.

The term "fetal size abnormality (FSA)" refers collectively to fetuses that are small or large for gestational age. The two main classes of FSA are discussed below.

Fetal growth restriction

Fetal growth restriction (FGR), also referred to as intrauterine growth restriction (IUGR), occurs in approximately 3% of all pregnancies (the estimated incidence varies depending on the definition employed), and is a condition in which the fetus is smaller than expected for the number of weeks of gestation. FGR is thought to be the major single cause of stillbirth and is associated with an increased risk of neonatal morbidity and mortality, poor health and educational achievement in childhood, and an increased risk of developing diseases and disorders such as coronary heart disease, stroke, hypertension and type 2 diabetes in later life. One third of all stillbirths occur at term and infants with a birth weight less than the 3^rd percentile at term have an eight-fold higher risk of antepartum stillbirth.

A large proportion of adverse events associated with FGR occur in the absence of known risk factors and this has motivated a substantial body of research into the development of diagnostic methods for FGR. False positive diagnosis of FGR has the potential to cause considerable harm by iatrogenic prematurity because the primary intervention for FGR is early delivery. The risk of harm is heightened when delivery is preterm (i.e. prior to 37 weeks).

The most promising potential approach to screening for FGR is universal ultrasound, but ultrasound has not been shown to result in better outcomes for mother or baby, potentially due to the high false positive rate of diagnosis. The limitations of ultrasound in the context of FGR are particularly problematic because ultrasound is even less effective as a screening test for term FGR than preterm FGR. Consequently, the primary method of screening for FGR in low risk women in the USA, UK and many other countries remains clinical examination, such as measurement of the symphyseal-fundal height followed by ultrasound if fundal height is smaller than expected.

FGR has pathophysiological features in common with preeclampsia and it has been suggested that diagnostic methods for preeclampsia might be useful in the detection of FGR. The soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth actor (PIGF) ratio is increasingly used to rule in or rule out the diagnosis of preeclampsia in pregnant women where the condition is clinically suspected. Although the sFlt-l:PIGF ratio has been shown to have some predictive power in detecting FGR, it exhibits an undesirably high false positive rate. When an elevated ratio was defined as >38, measurement at 36wkGA had a sensitivity of 53% to detect FGR but a specificity of only 86%. Women who have previously suffered from FGR are at a higher risk of developing FGR during subsequent pregnancies and therefore receive enhanced monitoring of fetal development throughout their pregnancy. It is especially difficult to both predict and prevent adverse pregnancy outcomes in nulliparous women due to a lack of previous pregnancy information to help guide risk assessment. Consequently, nulliparous women have a yet heightened need for diagnostic tests for FGR.

Large for gestational age

A large for gestational age (LGA) infant is defined as one where either the estimated fetal weight or birth weight is higher than a given threshold percentile for the given week of pregnancy. Thresholds employed include the 90th, 95th and 97th and the threshold employed determines the prevalence (~10%, ~5% and ~3%, respectively). Macrosomia is defined as a baby where the birth weight is above a given extreme value of weight, irrespective of gestational age - thresholds employed include >4000g (used e.g. in the USA) and >4500g (used e.g. in the UK). LGA and macrosomia are both clearly related but distinct entities. Another major determinant of birth weight is the gestational age of the fetus, with the baby getting progressively bigger as pregnancy becomes more advanced. At preterm gestational ages, few LGA babies may be macrosomic. However, at term, many LGA babies will also be macrosomic.

Those with LGA are at higher risk of morbidity, including shoulder dystocia (delay in delivering the baby's body after delivery of the head affecting ~1% of vaginal deliveries) and brachial plexus injury (damage to nerves arising from the neck typically caused by traction to effect delivery when shoulder dystocia occurs). Those with LGA are also at higher risk of death including both stillbirth and death in the neonatal period. LGA is commonly associated with an increased risk of requiring delivery by emergency caesarean section.

Ultrasound can be used as a means to identify LGA, but the inventors believe that ultrasound can yield false negative results for more than 50% of large babies. Fetal body parts (e.g. head, abdomen and thigh bone) are measured using electronic callipers and an estimated fetal weight (EFW) is calculated using a statistical model.

When LGA has been diagnosed, two common candidate interventions include planned caesarean delivery, which may help prevent the risk of birth injury, and early induction of labour, which may reduce birth weight by abbreviating the duration of pregnancy. A randomised controlled trial demonstrated reduced rates of significant shoulder dystocia following induction of labour at 37-38 wkGA for suspected LGA compared with expectant management (from 4% to 1%, respectively). That study employed women who had ultrasound scans for clinically suspected LGA and used an estimated fetal weight threshold of >95th percentile for gestational age (hence the higher background risk of shoulder dystocia).

However, ultrasonic EFW is associated with high proportions of false positives and false negatives for LGA. A false positive diagnosis of LGA is associated with increased rates of unnecessary intervention. Specifically, babies which ultimately had a normal birth weight were more likely to be delivered by emergency caesarean section during labour if there was a false positive diagnosis of LGA. Conversely, a false negative diagnosis may lead to a baby experiencing birth injury through shoulder dystocia which could have been prevented by early induction of labour.

Currently, clinical guidelines in the UK and the US recommend that women should not be routinely screened using ultrasound in the last third of pregnancy, as there is no clear evidence of benefit from a meta-analysis of randomized controlled trials. False positive ultrasonic diagnoses have the potential to cause harm through unnecessary intervention. However, the UK Guidelines recommend further research on the diagnostic effectiveness of universal ultrasound.

There is an urgent and unmet need for sensitive and specific methods to identify subjects having an increased risk of FSA. There is a particular need for a sensitive and specific methods to identify an increased risk of FSA in women who are nulliparious and/or women who are at term. There is likewise an urgent and unmet need for sensitive and specific methods to identify subjects having an increased risk of FGR. There is a particular need for sensitive and specific methods to identify an increased risk of FGR in women who are nulliparious and/or women who are at term.

There is likewise an urgent and unmet need for sensitive and specific methods to identify subjects having an increased risk of LGA. There is a particular need for sensitive and specific methods to identify an increased risk of LGA in women who are nulliparious and/or women who are at term.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the above needs by providing methods of screening for risk of FSA, and metabolites for use in said methods.

The present invention also addresses one or more of the above needs by providing methods of screening for risk of FGR, and metabolites for use in said methods. Methods of the invention achieve superior specificity (i.e. lower false positive rate) than existing methods for identifying increased risk of FGR using analysis of maternal blood, including the currently used method based on the sFIt- 1:PIGF ratio.

The present invention addresses one or more of the above needs by providing methods of screening for risk of LGA, and metabolites for use in said methods. Methods of the invention overcome limitations of existing methods for identifying increased risk of LGA, such as ultrasound.

The present invention is based upon the surprising discovery of nine metabolites that are not only predictive of an increased risk of FSA, but are independently predictive of FSA. Moreover, analysis of the directional change in the level of said independently predictive metabolites, as compared to controls, advantageously achieves identification of an increased risk of FGR or an increased risk of LGA. The identification of independently predictive metabolites provides significant advantages because each of the identified metabolites provides a powerful contribution to the diagnosis of increased risk, which can far exceed the diagnostic capabilities achievable through the analysis of metabolites that are not independently predictive. The invention achieves advantageously improved diagnosis of an increased risk of FSA through the identification of a difference in the level of two or more of said newly-identified independently predictive metabolites. Moreover, the directionality of the change in the level of said two or more independently predictive metabolites can advantageously be used to distinguish between an increased risk of FGR and an increased risk of LGA.

Through the analysis of the newly-identified independently predictive metabolites, methods of the invention offer several key advantages over FSA diagnostic methods currently employed in the clinic. For example, in the context of FGR, such benefits of the invention include: (1) higher specificity than the sFlt-1: PIGF ratio for a given sensitivity; (2) improved suitability for use in the diagnosis of FGR in nulliparous women; and (3) superior diagnosis of FGR at term. Similar advantages are achieved by the invention in comparison to LGA diagnostic methods currently employed in the clinic.

The inventors first sought to identify new metabolite markers for FGR. In an attempt to identify new metabolite markers for FGR, the inventors compared the level of >800 metabolites at various stages of pregnancy in women who delivered an FGR baby at term and those that did not. Diagnostic effectiveness requires large effect sizes and the inventors imposed a highly conservative significance threshold (P < 0.0005) to identify those metabolites which were likely to be diagnostically useful. This threshold was applied to metabolites which had already been selected as being in the top 100 candidate predictors, on the basis of differences in levels observed in measurements made earlier in the pregnancy. The conservative statistical approach selected by the inventors achieved the identification of nine metabolites that were not only predictive of an increased risk of FGR, but were independently predictive.

Methods of the invention are able to identify an increased risk of FGR with higher specificity than the sFlt-l:PIGF ratio. Increased specificity is highly advantageous in the clinic because it reduces the risk of causing harm by unnecessary interventions, such as early delivery. Increasing the accuracy of FGR diagnosis has the potential to significantly improve clinical outcomes for both the mother and child.

Advantageously, methods of the invention are suitable for use either alone or in combination with existing methods of diagnosing FSA. In this regard, the inventors found that by combining a method of the invention with an existing method of estimating fetal weight ( e.g . ultrasound), it was possible to significantly reduce the rate of false positive diagnosis of FGR from -14% (when using ultrasound alone), to -2% (when combining ultrasound with a method of the invention; see Table 5).

Methods of the invention also achieve improved sensitivity of diagnosis of FGR as compared to the sFlt-l:PIGF ratio which, when combined with estimation of fetal weight by ultrasound, only identified -21% of FGR cases. By contrast, methods of the invention successfully identified the majority of FGR cases.

As mentioned above, nulliparous women have the greatest need for diagnostically effective screening tests for FSA due to a lack of previous pregnancy information to help guide risk assessment. The inventors identified the metabolites of the invention in a cohort of nulliparous women, thereby ensuring that the metabolites are predictive of FGR irrespective of the existence of previous pregnancy information.

Methods that can reliably identify an increased risk of FSA, including term FGR, are particularly useful because adverse outcomes can be prevented by clinical intervention. The metabolites of the invention were identified in women who subsequently delivered an FGR baby at term, and so methods of the invention are ideally suited to the detection of term FGR.

Thus, not only is the method of the invention superior to existing methods of diagnosing FGR in terms of specificity, it can also successfully achieve these advantages in diagnosing term FGR (where adverse outcomes can be readily and safely prevented by early delivery), and in subjects who are nulliparous. The present invention is of considerable clinical significance. Unexpectedly, the inventors discovered that metabolites identified as being predictive of FGR are also predictive of LGA. Moreover, prediction of LGA according to the invention offers significant advantages over diagnostic methods in the art. Methods that can reliably identify an increased risk of LGA are particularly useful because adverse outcomes can be prevented by clinical intervention.

Advantageously, the metabolites of the invention are independently predictive of both types of FSA (i.e. FGR and LGA) and so detection of a change in the level of two or more of said metabolites may conveniently be used to identify FSA. Identification of FSA (or FGR or LGA more specifically) may be used to inform and optimise subject management. For example, monitoring of the subject may be enhanced e.g. by increased frequency of fetal assessment by ultrasound; and/or the duration of pregnancy may be abbreviated; and/or the mode of delivery may be changed to caesarean section.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Distribution of P values from the composite Chi-squared test (two-sided) for the measurements at 20/28 weeks of gestational age (wkGA). The P values of 829 metabolites with a known structural identity were calculated from the test for interaction between term FGR and gestational age.

Figure 2. Mean (95%CI) of four selected metabolites by term FGR case status in the Pregnancy Outcome Prediction (POP) study.

Figure 3. ROC curve analysis of the ratio of two products of metabolites (predictive ratio) and the sFLTl:PIGF ratio at 36wkGA in relation to term FGR (162 cases and 275 controls). Area under the curve (AUC) (95%CI) was 0.778 (0.732 to 0.824) for the predictive ratio and 0.636 (0.581 to 0.691) for the sFLTl:PIGF ratio.

Figure 4. ROC curve analysis of the ratio of two products of metabolites (metabolite ratio) at ~24-28wkGA in relation to FGR (birthweight <3rd percentile) in the POP study (AUC=0.72, 95% Cl:

0.67 to 0.77). Figure 5. Odds ratios (95% confidence intervals) of the measurements of metabolite ratio at 36 wkGA in relation to term FGR (n=175) by maternal characteristics in the case cohort sample (n=299 term controls without FGR). Abbreviations: EFW denotes estimated fetal weight, FGR denotes fetal growth restriction, wkGA denotes weeks of gestational age.

Figure 6. Odds ratios (95% confidence intervals) of the measurements of (A) metabolite ratio and (B) sFlt-l:PIGF ratio at 36 wkGA in relation to term FGR (n=175) by phenotype in the case cohort sample (n=299 term controls without FGR). Abbreviations: BW denotes birth weight, ACGV denotes abdominal circumference growth velocity, FGR denotes fetal growth restriction, wkGA denotes weeks of gestational age.

Figure 7. Metabolite ratio developed for FGR and birth weight as a continuous outcome. Customized birthweight z score by the z score of the log-transformed metabolite ratio at 36 wkGA developed for FGR in healthy women from the comparator group who delivered at term (n=272). The red line describes the linear association (y = -0.06 - 0.37 x) and the black lines represent the lower and upper bounds of the 95% confidence intervals. Customized birthweight percentiles (GROW bulk calculator, version 6.7.8.1, Perinatal Institute, Birmingham, UK) were turned into z scores using the invnormal function in Stata. GROW, Gestation-Related Optimal Weight; wkGA, weeks of gestational age.

DETAILED DESCRIPTION OF THE INVENTION

Early and accurate diagnosis of an increased risk of FSA is essential to allow appropriate antenatal monitoring and intervention plans to be put in place. This is particularly critical for term FGR and for subjects who are nulliparous. Accurate diagnosis of FSA (or of FGR or LGA more specifically), has the potential to reduce the rate of adverse outcomes associated with FSA for both mother and child.

As used herein, FGR is defined as an infant with a birth weight <3^rd percentile. FGR also includes infants born with a birth weight <10^th percentile when coupled with evidence ( e.g . ultrasound evidence) of reduced fetal growth velocity. "Term FGR" refers to a fetus that is affected by growth restriction and is born at term.

As used herein, LGA is defined as an infant with a birth weight >90^th percentile.

As used herein, FSA is defined as an infant with a birth weight <10^th percentile and >90^th percentile.

In an attempt to identify new metabolite markers for FGR, the inventors compared the level of >800 metabolites at various stages of pregnancy in women who delivered an FGR baby at term and those that did not. Initially, 100 metabolites were identified with levels that differed between the two groups of women assessing blood samples obtained at around 12, 20 and 28 weeks of gestational age. The inventors imposed a highly conservative significance threshold (P < 0.0005) to identify which of these metabolites were likely to have the greatest clinical relevance using a fourth blood sample from the women, obtained at around 36 weeks of gestational age. This conservative approach identified 22 metabolites with levels in the fourth blood sample that differed significantly between women who delivered an FGR baby and those that did not.

Independently predictive factors can improve considerably the predictive power of diagnostic methods because, when combined, they each provide a distinct (and substantially non-overlapping) contribution to the overall diagnosis. The identification of independently predictive metabolites is therefore highly desirable. To help identify metabolites that are independently predictive of FGR, the inventors used a forward stepwise logistic regression approach which included maternal age, BMI and the sFlt-l:PIGF ratio in addition to the 22 metabolites. Unexpectedly, this approach achieved the identification of nine metabolites that are independently predictive of FGR.

Each of the nine independently predictive metabolites identified by the inventors are significantly associated with FGR. The nine newly-identified metabolites include:

(a) l-(l-enyl-stearoyl)-2-oleoyl-GPC

(b) 1,5-anhydroglucitol (c) 5alpha-androstan-3alpha,17alpha-diol disulfate

(d) Nl,N12-diacetylspermine

(e) 4-androsten-3beta,17beta-diol monosulfate (2)

(f) Acisoga

(g) Estriol 3-sulfate

(h) 4-cholesten-3-one

(i) Cotinine N-oxide

Of the nine metabolites, the following are positively associated with the risk of FGR: l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide. Of the nine metabolites, the following are negatively associated with the risk of FGR: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

The invention provides use of two or more metabolites selected from the list consisting of: l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide, for the identification of an increased risk of FGR in a subject.

Methods of the invention provide superior diagnosis of FGR, as compared to diagnostic methods currently employed.

Unexpectedly, the inventors discovered that metabolites identified as being independently predictive of FGR are also predictive of LGA. Metabolites identified as being positively associated with FGR were observed to be negatively associated with LGA. Conversely, metabolites identified as being negatively associated with FGR were observed to be positively associated with LGA. Of the nine metabolites, the following are negatively associated with the risk of LGA: l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide. Of the nine metabolites, the following are positively associated with the risk of LGA: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

The invention provides use of two or more metabolites selected from the list consisting of: l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide, for the identification of an increased risk of LGA in a subject.

Methods of the invention provide superior diagnosis of LGA, as compared to diagnostic methods currently employed.

The invention provides a method of screening for risk of FSA in a subject, the method comprising: (a) quantifying in a biological sample obtained from the subject the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; and (b) comparing the level of said two or more metabolites in the biological sample with reference level of said two or more metabolites; wherein a difference between the level of said two or more metabolites in the biological sample and the reference level of said two or more metabolites is indicative of an increased risk of FSA in the subject.

The invention provides a method of screening for risk of FGR in a subject, the method comprising: (a) quantifying in a biological sample obtained from the subject the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; and (b) comparing the level of said two or more metabolites in the biological sample with reference level of said two or more metabolites; wherein:

(i) an increase in the level of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in the biological sample as compared to the reference; and/or

(ii) a decrease in the level of one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate 4-cholesten-3-one in the biological sample as compared to the reference; is indicative of an increased risk of FGR in the subject.

In one embodiment, said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC. A higher level of l-(l-enyl-stearoyl)-2-oleoyl-GPC in the test subject compared to a reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC is indicative of an increased risk of FGR. In one embodiment, at least a 5% increase in the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC compared to a reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC compared to a reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise 1,5-anhydroglucitol. A higher level of 1,5-anhydroglucitol in the test subject compared to a reference level of 1,5-anhydroglucitol is indicative of an increased risk of FGR. In one embodiment, at least a 5% increase in the level of 1,5- anhydroglucitol compared to a reference level of 1,5-anhydroglucitol is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of 1,5-anhydroglucitol compared to a reference level of 1,5-anhydroglucitol is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise 5alpha-androstan-3alpha,17alpha-diol disulfate. A lower level of 5alpha-androstan-3alpha,17alpha-diol disulfate in the test subject compared to a reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate is indicative of an increased risk of FGR. In one embodiment, at least a 5% decrease in the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate compared to a reference level of 5alpha-androstan-3alpha,17alpha- diol disulfate is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of 5alpha-androstan-3alpha,17alpha-diol disulfate compared to a reference level of 5alpha- androstan-3alpha,17alpha-diol disulfate is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise Nl,N12-diacetylspermine. A lower level of Nl,N12-diacetylspermine in the test subject compared to a reference level of N1,N12- diacetylspermine is indicative of an increased risk of FGR. In one embodiment, at least a 5% decrease in the level of Nl,N12-diacetylspermine compared to a reference level of Nl,N12-diacetylspermine is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of N1,N12- diacetylspermine compared to a reference level of Nl,N12-diacetylspermine is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise 4-androsten-3beta,17beta-diol monosulfate (2). A higher level of 4-androsten-3beta,17beta-diol monosulfate (2) in the test subject compared to a reference level of 4-androsten-3beta,17beta-diol monosulfate (2) is indicative of an increased risk of FGR. In one embodiment, at least a 5% increase in the level of 4-androsten-

3beta,17beta-diol monosulfate (2) compared to a reference level of 4-androsten-3beta,17beta-diol monosulfate (2) is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of 4-androsten-3beta,17beta-diol monosulfate (2) compared to a reference level of 4- androsten-3beta,17beta-diol monosulfate (2) is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise Acisoga. A higher level of Acisoga in the test subject compared to a reference level of Acisoga is indicative of an increased risk of FGR. In one embodiment, at least a 5% increase in the level of Acisoga compared to a reference level of Acisoga is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of Acisoga compared to a reference level of Acisoga is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise Estriol 3-sulfate. A lower level of Estriol 3-sulfate in the test subject compared to a reference level of Estriol 3-sulfate is indicative of an increased risk of FGR. In one embodiment, at least a 5% decrease in the level of Estriol 3-sulfate compared to a reference level of Estriol 3-sulfate is indicative of an increased risk of FGR. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of Estriol 3-sulfate compared to a reference level of Estriol 3-sulfate is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise 4-cholesten-3-one. A lower level of 4- cholesten-3-one in the test subject compared to a reference level of 4-cholesten-3-one is indicative of an increased risk of FGR. In one embodiment, at least a 5% decrease in the level of 4-cholesten-3- one compared to a reference level of 4-cholesten-3-one is indicative of an increased risk of FGR. In one embodiment, at least at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of 4-cholesten-3-one compared to a reference level of 4-cholesten-3-one is indicative of an increased risk of FGR.

In one embodiment, said two or more metabolites comprise cotinine N-oxide. A higher level of cotinine N-oxide in the test subject compared to a reference level of cotinine N-oxide is indicative of an increased risk of FGR. In one embodiment, at least a 5% increase in the level of cotinine N-oxide compared to a reference level of cotinine N-oxide is indicative of an increased risk of FGR. In one embodiment at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of cotinine N-oxide compared to a reference level of cotinine N-oxide is indicative of an increased risk of FGR.

In one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more metabolites that are positively associated with the risk of having FGR. Thus, in one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide.

In one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more metabolites that are negatively associated with the risk of having FGR. Thus, in one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

The invention provides a method of screening for risk of LGA in a subject, the method comprising: (a) quantifying in a biological sample obtained from the subject the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; and (b) comparing the level of said two or more metabolites in the biological sample with reference level of said two or more metabolites; wherein:

(i) a decrease in the level of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in the biological sample as compared to the reference; and/or

(ii) an increase in the level of one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate 4-cholesten-3-one in the biological sample as compared to the reference; is indicative of an increased risk of LGA in the subject.

In one embodiment, said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC. A lower level of l-(l-enyl-stearoyl)-2-oleoyl-GPC in the test subject compared to a reference level of 1- (l-enyl-stearoyl)-2-oleoyl-GPC is indicative of an increased risk of LGA. In one embodiment, at least a 5% decrease in the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC compared to a reference level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC compared to a reference level of l-(l-enyl- stearoyl)-2-oleoyl-GPC is indicative of an increased risk of LGA.

In one embodiment, said two or more metabolites comprise 1,5-anhydroglucitol. A lower level of 1,5-anhydroglucitol in the test subject compared to a reference level of 1,5-anhydroglucitol is indicative of an increased risk of LGA. In one embodiment, at least a 5% decrease in the level of 1,5- anhydroglucitol compared to a reference level of 1,5-anhydroglucitol is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of 1,5-anhydroglucitol compared to a reference level of 1,5-anhydroglucitol is indicative of an increased risk of LGA.

In one embodiment, said two or more metabolites comprise 5alpha-androstan-3alpha,17alpha-diol disulfate. A higher level of 5alpha-androstan-3alpha,17alpha-diol disulfate in the test subject compared to a reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate is indicative of an increased risk of LGA. In one embodiment, at least a 5% increase in the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate compared to a reference level of 5alpha-androstan-3alpha,17alpha- diol disulfate is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a

15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of 5alpha-androstan-3alpha,17alpha-diol disulfate compared to a reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate is indicative of an increased risk of LGA.

In one embodiment, said two or more metabolites comprise Nl,N12-diacetylspermine. A higher level of Nl,N12-diacetylspermine in the test subject compared to a reference level of N1,N12- diacetylspermine is indicative of an increased risk of LGA. In one embodiment, at least a 5% increase in the level of Nl,N12-diacetylspermine compared to a reference level of Nl,N12-diacetylspermine is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of

Nl,N12-diacetylspermine compared to a reference level of Nl,N12-diacetylspermine is indicative of an increased risk of LGA. In one embodiment, said two or more metabolites comprise 4-androsten-3beta,17beta-diol monosulfate (2). A lower level of 4-androsten-3beta,17beta-diol monosulfate (2) in the test subject compared to a reference level of 4-androsten-3beta,17beta-diol monosulfate (2) is indicative of an increased risk of LGA. In one embodiment, at least a 5% decrease in the level of 4-androsten- 3beta,17beta-diol monosulfate (2) compared to a reference level of 4-androsten-3beta,17beta-diol monosulfate (2) is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of 4-androsten-3beta,17beta-diol monosulfate (2) compared to a reference level of 4- androsten-3beta,17beta-diol monosulfate (2) is indicative of an increased risk of LGA.

In one embodiment, said two or more metabolites comprise Acisoga. A lower level of Acisoga in the test subject compared to a reference level of Acisoga is indicative of an increased risk of LGA. In one embodiment, at least a 5% decrease in the level of Acisoga compared to a reference level of Acisoga is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of Acisoga compared to a reference level of Acisoga is indicative of an increased risk of LGA.

In one embodiment, said two or more metabolites comprise Estriol 3-sulfate. A higher level of Estriol 3-sulfate in the test subject compared to a reference level of Estriol 3-sulfate is indicative of an increased risk of LGA. In one embodiment, at least a 5% increase in the level of Estriol 3-sulfate compared to a reference level of Estriol 3-sulfate is indicative of an increased risk of LGA. In one embodiment, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of Estriol 3-sulfate compared to a reference level of Estriol 3-sulfate is indicative of an increased risk of LGA. In one embodiment, said two or more metabolites comprise 4-cholesten-3-one. A higher level of 4- cholesten-3-one in the test subject compared to a reference level of 4-cholesten-3-one is indicative of an increased risk of LGA. In one embodiment, at least a 5% increase in the level of 4-cholesten-3- one compared to a reference level of 4-cholesten-3-one is indicative of an increased risk of LGA. In one embodiment, at least at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 100% increase in the level of 4-cholesten-3-one compared to a reference level of 4-cholesten-3-one is indicative of an increased risk of LGA.

In one embodiment, said two or more metabolites comprise cotinine N-oxide. A lower level of cotinine N-oxide in the test subject compared to a reference level of cotinine N-oxide is indicative of an increased risk of LGA. In one embodiment, at least a 5% decrease in the level of cotinine N-oxide compared to a reference level of cotinine N-oxide is indicative of an increased risk of LGA. In one embodiment at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or a 100% decrease in the level of cotinine N-oxide compared to a reference level of cotinine N-oxide is indicative of an increased risk of LGA.

In one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more metabolites that are negatively associated with the risk of having LGA. Thus, in one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide.

In one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more metabolites that are positively associated with the risk of having LGA. Thus, in one embodiment, the level of said two or more metabolites corresponds to the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

In one embodiment, the combination of the level of said two or more metabolites corresponds to the sum of the level of said two or more metabolites.

In one embodiment, the combination of the level of said two or more metabolites corresponds to the product of the level of said two or more metabolites.

In one embodiment, the combination of the level of said two or more metabolites corresponds to the result of any mathematical operation applied to the level of said two or more metabolites, wherein application of said mathematical operation yields a value greater than the individual values entered into the operation, e.g. addition, multiplication or exponentiation.

In one embodiment, the combination of the level of said two or more metabolites corresponds to any combination of the sum, the product, or any mathematical operation applied to the level of said two or more metabolites, wherein application of said mathematical operation yields a value greater than the individual values entered into the operation. For example, any combination of addition, multiplication and/or exponentiation.

In one embodiment, the method comprises applying a mathematical model to the level of said two or more metabolites. In one embodiment, said mathematical model is a statistical model. In one embodiment, said statistical model comprises logistic regression. In one embodiment, the statistical model comprises a multivariate model, optionally a multivariate logistic regression model. Suitable mathematical models and methods of applying mathematical models are well known in the art.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; (b) comparing the combination of the level of said two or more metabolites in the biological sample with the combination of the reference level of said two or more metabolites; wherein a difference between the combination of the level of said two or more metabolites and the combination of the reference level of said two or more metabolites is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; (b) comparing the combination of the level of said two or more metabolites in the biological sample with the combination of the reference level of said two or more metabolites; wherein a greater combination of the level of said two or more metabolites than the combination of the reference level of said two or more metabolites is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; (b) comparing the combination of the level of said two or more metabolites in the biological sample with the combination of the reference level of said two or more metabolites; wherein a lower combination of the level of said two or more metabolites than the combination of the reference level of said two or more metabolites is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises comparing the combination of the level of any two, any three, any four or all five metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide to the combination of the reference levels of said metabolites.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) comparing the combination of the level of said two or more metabolites in the biological sample with the combination of the reference level of said two or more metabolites; wherein a difference between the combination of the level of said two or more metabolites and the combination of the reference level of said two or more metabolites is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) comparing the combination of the level of said two or more metabolites in the biological sample with the combination of the reference level of said two or more metabolites; wherein a lower combination of the level of said two or more metabolites than the combination of the reference level of said two or more metabolites is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) comparing the combination of the level of said two or more metabolites in the biological sample with the combination of the reference level of said two or more metabolites; wherein a greater combination of the level of said two or more metabolites than the combination of the reference level of said two or more metabolites is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises comparing the combination of the level of any two, any three, or all four metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one to the combination of the reference levels of said metabolites.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol; (b) comparing the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample with the combination of the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the reference level of 1,5-anhydroglucitol; wherein a difference between the combination of the level 1- (l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol and the combination of the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the reference level of 1,5-anhydroglucitol is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol; (b) comparing the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample with the combination of the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the reference level of 1,5-anhydroglucitol; wherein a greater combination of the level l-(l-enyl- stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol than the combination of the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the reference level of 1,5-anhydroglucitol is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol; (b) comparing the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample with the combination of the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the reference level of 1,5-anhydroglucitol; wherein a lower combination of the level l-(l-enyl-stearoyl)- 2-oleoyl-GPC and the level of 1,5-anhydroglucitol than the combination of the reference level of 1- (l-enyl-stearoyl)-2-oleoyl-GPC and the reference level of 1,5-anhydroglucitol is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) comparing the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample with the combination of the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the reference level of N1,N12- diacetylspermine; wherein a difference between the combination of the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine and the combination of the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the reference level of N1,N12- diacetylspermine is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) comparing the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample with the combination of the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the reference level of N1,N12- diacetylspermine; wherein a lower combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine than the combination of the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the reference level of N1,N12- diacetylspermine is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) comparing the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample with the combination of the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the reference level of N1,N12- diacetylspermine; wherein a greater combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine than the combination of the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the reference level of N1,N12- diacetylspermine is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of one metabolite selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one metabolite selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of one metabolite selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one metabolite selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of one metabolite selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol,

4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one metabolite selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

The "reference metabolite ratio" is determined based upon reference levels of the same metabolites as form the basis of the subject metabolite ratio.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of one metabolite selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one metabolite selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of one metabolite selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one metabolite selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of one metabolite selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one metabolite selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject. In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and two or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject. In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of 5alpha-androstan-

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1,5- anhydroglucitol, the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1,5- anhydroglucitol, the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1,5- anhydroglucitol, the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the combination of the level of estriol 3-sulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the product of the level of 1- (l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the product of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the product of the level of 1- (l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the product of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the corresponding reference metabolite ratio is indicative of an increased risk of FGR in the subject. In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the product of the level of 1- (l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; divided by (ii) the product of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the corresponding reference metabolite ratio is indicative of an increased risk of LGA in the subject.

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the product of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; divided by (ii) the product of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the product of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; divided by (ii) the product of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a lower subject metabolite ratio than the corresponding reference metabolite ratio is indicative of an increased risk of FGR in the subject.

In one embodiment, the method comprises: (a) quantifying in the biological sample the level of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5-anhydroglucitol, the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine; (b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the product of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; divided by (ii) the product of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a greater subject metabolite ratio than the corresponding reference metabolite ratio is indicative of an increased risk of LGA in the subject.

In one embodiment, the method comprises comparing the ratio of the combination of the level of any two, any three, any four or all five metabolites selected from the list consisting of: l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide divided by the combination of the level of any two, any three, or all four of the metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one to a reference ratio of said metabolites.

In one embodiment, the method comprises comparing the ratio of the combination of the level of any two, any three, or all four of the metabolites selected from the list consisting of: 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten- 3-one divided by the combination of the level of any two, any three, any four or all five metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide to a reference ratio of said metabolites. Wherein a metabolite ratio is the ratio of: (i) level(s) or a combination of levels of metabolites that are positively associated with an increased risk of FGR, divided by (ii) level(s) or a combination of levels of metabolites that are negatively associated with an increased risk of FGR, the value of the subject metabolite ratio in patients with an increased risk of FGR, is typically greater than the corresponding reference metabolite ratio.

Wherein a metabolite ratio is the ratio of: (i) level(s) or a combination of levels of metabolites that are negatively associated with an increased risk of FGR, divided by (ii) level(s) or a combination of levels of metabolites that are positively associated with an increased risk of FGR, the value of the subject metabolite ratio in patients with an increased risk of FGR, is typically lower than the corresponding reference metabolite ratio.

Wherein a metabolite ratio is the ratio of: (i) level(s) or a combination of levels of metabolites that are positively associated with an increased risk of LGA, divided by (ii) level(s) or a combination of levels of metabolites that are negatively associated with an increased risk of LGA, the value of the subject metabolite ratio in patients with an increased risk of LGA, is typically greater than the corresponding reference metabolite ratio.

Wherein a metabolite ratio is the ratio of: (i) level(s) or a combination of levels of metabolites that are negatively associated with an increased risk of LGA, divided by (ii) level(s) or a combination of levels of metabolites that are positively associated with an increased risk of LGA, the value of the subject metabolite ratio in patients with an increased risk of LGA, is typically lower than the corresponding reference metabolite ratio.

The metabolites of the invention are individually predictive of FGR. Because the metabolites of the invention are also independently predictive of FGR, analysis of a combination of the level of metabolites of the invention provides even greater prediction of FGR, as compared to analysis of individual metabolites of the invention. For example, the product of l-(l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol is a superior predictor of FGR (AUC=0.70) than either of these metabolites separately (l-(l-enyl-stearoyl)-2-oleoyl-GPC, AUC=0.64 and 1,5-anhydroglucitol, AUC=0.65). Thus, in one embodiment, the method comprises quantifying the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol in a sample obtained from a subject; combining the levels of these two metabolites ( e.g . by multiplication); and comparing the combination of the level of these metabolites to the combination (e.g. the product) of reference levels of said metabolites. Levels of 1- (l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol are positively associated with FGR, and so a higher combination of levels in the test sample than the combination of reference levels is indicative of FGR.

Similarly the product of 5alpha-androstan-3alpha,17alpha-diol disulfate and N1,N12- diacetylspermine is a better predictor of FGR (product AUC=0.71) than either of these metabolites separately (5alpha-androstan-3alpha,17alpha-diol disulfate, AUC=0.69 and N1,N12- diacetylspermine, AUC=0.66). Thus, in one embodiment, the method comprises quantifying the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine in a sample obtained from a subject; combining the level of these two metabolites (e.g. by multiplication); and comparing the combination of the level of these metabolites to the combination (e.g. the product) of reference levels of said metabolites. Levels of 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine are negatively associated with FGR, and so a lower combination of levels in the test sample than the combination of reference levels is indicative of FGR.

The metabolites of the invention are individually predictive of LGA and so analysis of a combination of the level of metabolites of the invention provides even greater prediction of LGA, as compared to analysis of individual metabolites of the invention. For example, the product of l-(l-enyl-stearoyl)-2- oleoyl-GPC and 1,5-anhydroglucitol is a superior predictor of LGA (AUC=0.82) than either of these metabolites separately (l-(l-enyl-stearoyl)-2-oleoyl-GPC, AUC= 0.80 and 1,5-anhydroglucitol, AUC= 0.74). Thus, in one embodiment, the method comprises quantifying the level of l-(l-enyl-stearoyl)-

2-oleoyl-GPC and 1,5-anhydroglucitol in a sample obtained from a subject; combining the levels of these two metabolites ( e.g . by multiplication); and comparing the combination of the level of these metabolites to the combination (e.g. the product) of reference levels of said metabolites. Levels of 1- (l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol are negatively associated with LGA, and so a lower combination of levels in the test sample than the combination of reference levels is indicative of LGA.

The product of 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine is also a strong predictor of LGA (product AUC=0.66). Thus, in one embodiment, the method comprises quantifying the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and N1,N12- diacetylspermine in a sample obtained from a subject; combining the level of these two metabolites (e.g. by multiplication); and comparing the combination of the level of these metabolites to the combination (e.g. the product) of reference levels of said metabolites. Levels of 5alpha-androstan- 3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine are positively associated with LGA, and so a higher combination of levels in the test sample than the combination of reference levels is indicative of LGA.

Whilst methods of the invention may be performed using simple analysis (e.g. by simple combination of metabolite levels), useful diagnosis of FSA (or FGR or LGA more specifically) may also be achieved using more complex statistical methods. For example, a common approach in the field of diagnostics is to employ complex multivariate statistical models to link the presence and/or quantity of metabolites to a particular clinical outcome. Thus, in one embodiment, the method comprises using a multivariate model to identify an increased risk of FSA (or FGR or LGA more specifically). In one embodiment, the method comprises using a regression model. In one embodiment, the method comprises using a multivariate logistic regression model. A multivariate logistic regression model combining the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine was able to diagnose FGR with an AUC of 0.783. The inventors achieved a similar level of predictive accuracy when applying a simple combination of levels of the metabolites, as when these levels were subjected to the more complex multivariate logistic regression model. For example, when diagnosing FGR, the metabolite ratio of the product of l-(l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol divided by the product of 5alpha- androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine, achieved a similar level of predictive accuracy as the multivariate logistic regression model (metabolite ratio AUC=0.778; regression model AUC=0.783, P> 0.5).

The skilled person would understand that for the metabolite ratios disclosed herein, substituting the numerator and denominator would achieve substantially the same predictive accuracy.

The skilled person would also understand that when calculating a ratio, metabolites that form the numerator will typically have a different direction of association than the metabolites forming the denominator. That is, when the numerator is formed of the level of, or the combination of levels of two or more of, l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta- diol monosulfate (2), Acisoga and cotinine N-oxide; the denominator will be formed of the level of, or the combination of the level of two or more of, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one, or vice versa.

A further advantage provided by the claimed invention is that clinically desirable predictive performance is also achieved at relatively early stages in pregnancy, which cannot be achieved when using current approaches, as evidenced by the lack of improved outcomes provided by universal ultrasound. The early stage predictive capability achieved by the present invention is illustrated in Figure 4 which demonstrates that the above metabolite ratio also achieves good predictive performance of term FGR when using samples obtained at 24-28 wkGA (AUC=0.72). An early indication that a subject is at risk of developing FGR allows for increased monitoring of the pregnancy and fetal growth so that potential complications can be identified early and managed to help reduce the risk of adverse outcomes. The term "diagnosis of FSA" or "FSA diagnosis" as used herein encompasses identification, confirmation, and/or characterisation of an increased risk of having a baby affected by FSA, as compared to control. A subject diagnosed as having FSA is a subject identified by a method of the invention as having an increased risk of having an FSA baby. "Predisposition" to FSA refers to a subject who does not currently present with symptoms related to FSA, but is at an increased risk of having an FSA baby, e.g. at term. "FSA occurring at term" as used herein is a fetus affected by FSA that is born at term.

The term "diagnosis of FGR" or "FGR diagnosis" as used herein encompasses identification, confirmation, and/or characterisation of an increased risk of having a baby affected by FGR, as compared to control. A subject diagnosed as having FGR is a subject identified by a method of the invention as having an increased risk of having an FGR baby. "Predisposition" to FGR refers to a subject who does not currently present with symptoms related to FGR, but is at an increased risk of having an FGR baby, e.g. at term. "FGR occurring at term" as used herein is a fetus affected by FGR that is born at term.

The term "diagnosis of LGA" or "LGA diagnosis" as used herein encompasses identification, confirmation, and/or characterisation of an increased risk of having a baby affected by LGA, as compared to control. A subject diagnosed as having LGA is a subject identified by a method of the invention as having an increased risk of having an LGA baby. "Predisposition" to LGA refers to a subject who does not currently present with symptoms related to LGA, but is at an increased risk of having an LGA baby, e.g. at term. "LGA occurring at term" as used herein is a fetus affected by LGA that is born at term.

The present invention provides for improved clinical outcomes with the potential for early, accurate diagnosis of FSA (or FGR or LGA more specifically), allowing rapid identification of the most appropriate monitoring and treatment plans and the targeting of interventions to women who are at high risk of complications. The method of the invention also reduces the risk of unnecessary exposure to harmful interventions. The terms "treating FSA", "treating FGR" and "treating LGA" as used herein, refer to the avoidance or prevention of adverse outcomes associated with FSA, FGR and LGA. For example, an FGR baby is more likely to become severely asphyxiated during labour, and interventions to deliver the baby by pre-labour C-section avoids this risk. The phrase "indicative of FSA", "indicative of FGR" and "indicative of LGA" as used herein, mean indicative of an increased risk of having such a baby.

Methods of the invention may further comprise performing one or more additional, different tests to confirm or exclude diagnosis of FSA (or FGR or LGA more specifically), and/or to further characterise a condition. In one embodiment, the method further comprises estimating fetal weight by fetal ultrasound. In one embodiment, the method further comprises estimating fetal weight by assessing fundal height. In one embodiment, the method further comprises assessing maternal, fetal or umbilical blood flow by Doppler ultrasound. In one embodiment, the method further comprises diagnosing preeclampsia, or a predisposition thereto, by measurement of the sFlt-l:PIGF ratio.

In one embodiment, upon identifying a subject as having an increased risk of FSA (or FGR or LGA more specifically), by a method of the invention, the method further comprises enhanced monitoring of the subject, e.g. by increased frequency of fetal assessment by ultrasound. Enhanced monitoring of the subject may involve increased frequency of fetal assessment by ultrasound as compared to the frequency of ultrasound that is typically employed when FSA (or FGR or LGA more specifically) is not suspected. Increased frequency of fetal assessment by ultrasound may involve performing ultrasound to estimate fetal weight at eight-week intervals, six-week intervals, four- week intervals, three-week intervals or two-week intervals, or more frequently, or at any other interval which represents an enhanced rate of assessment over what would have been recommended in the absence of the identification of the woman's increased risk of having a baby affected by FSA (or FGR or LGA more specifically). Enhanced monitoring of the subject may involve increased frequency of fundal height assessment, e.g. every two weeks, every week, every 4 days or more frequently.

In one embodiment, upon positive diagnosis of FSA (or FGR or LGA more specifically) by a method of the invention, the method further comprises administering a corticosteroid medicine to the subject.

In one embodiment, the method further comprises artificially inducing labour in a subject diagnosed as having FSA (or FGR or LGA more specifically). In one embodiment, artificially inducing labour comprises administering an induction agent to a subject, such as pharmacological or hormonal induction agent. Induction agents may include prostaglandins, e.g. PGE₂ or oxytocin. Appropriate dosages of induction agent are readily determined by the clinician. In one embodiment, artificially inducing labour comprises rupturing the amniotic sac. In one embodiment, artificially inducing labour comprises promoting ripening of the cervix through use of a medical device, e.g. a Foley catheter.

The invention also provides a method of treating FSA in a subject, the method comprising performing a caesarean section on a subject diagnosed by a method according to the invention as having an increased risk of FSA.

The invention also provides a method of treating FGR in a subject, the method comprising performing a caesarean section on a subject diagnosed by a method according to the invention as having an increased risk of FGR.

The invention also provides a method of treating LGA in a subject, the method comprising performing a caesarean section on a subject diagnosed by a method according to the invention as having an increased risk of LGA.

A "subject" according to the invention, also referred to as "patient" herein, is a pregnant woman. The subject can be a pregnant woman at any week of gestational age, preferably at least 12 weeks gestational age. In one embodiment, the subject is at least 20 weeks gestational age. In one embodiment, the subject is at least 28 weeks gestational age. In one embodiment, the subject is at least 36 weeks gestational age.

In one embodiment, the subject does not have diabetes.

A reference level is the level of metabolite(s) typically observed in a woman or women at the same week of gestational age as the subject, but who do not have or develop FSA (or FGR or LGA more specifically). Reference levels can also be the level of metabolite(s) in a randomly selected group of women where the FSA (or FGR or LGA more specifically) status is not known. In one embodiment, the reference level is a predetermined threshold level, wherein a subject metabolite level above or below said threshold value is indicative of an increased risk of FSA (or FGR or LGA more specifically). A reference metabolite ratio incorporates reference levels of metabolite(s). The reference level, or reference metabolite ratio, may comprise cut-off values or any other statistical attribute of the reference sample/group, such as a standard deviation from the mean levels of the metabolite(s).

No difference between the subject metabolite level or combination of levels and the reference level or combination of levels is indicative of no increased risk of FSA (or FGR or LGA more specifically).

No difference between the subject metabolite ratio and the reference metabolite ratio is indicative of no increased risk of FSA (or FGR or LGA more specifically).

Threshold values may be determined by statistical analysis of the reference sample/group to determine which level(s) represent a high likelihood that a subject is or is not at risk of having FSA (or FGR or LGA more specifically). In some embodiments, comparing the level of metabolite(s) in the sample obtained from the subject, or the subject metabolite ratio, is performed using other statistical methods. In related embodiments, comparing comprises logistic or linear regression. In other embodiments, comparing comprises computing an odds ratio.

References to metabolite levels also include references to a metabolite range. It will be appreciated that references herein to "difference between the level" refer to either a higher or lower level of the metabolite(s) in the test sample from the subject compared with the reference. It will also be appreciated that references herein to "difference between the subject metabolite ratio and the reference metabolite ratio" refer to either a higher or lower metabolite ratio in the subject sample compared with the reference metabolite ratio.

In one embodiment, the higher or lower level is a < 1 fold difference relative to the reference level, such as a fold difference of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 or any ranges therebetween. In one embodiment, the higher or lower level is between a 0.1 and 0.9 fold difference, such as between a 0.2 and 0.5 fold difference, relative to the reference level. In one embodiment, the higher or lower level is a > 1 fold difference relative to the reference level, such as a fold difference of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15 or 20 or any ranges therebetween. In one embodiment, the higher or lower level is between a 1 and 15 fold difference, such as between a 2 and 10 fold difference, relative to the reference level.

In one embodiment, the higher or lower metabolite ratio is a < 1 fold difference relative to the reference metabolite ratio, such as a fold difference of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 or any ranges therebetween. In one embodiment, the higher or lower metabolite ratio is a > 1 fold difference relative to the reference metabolite ratio, such as a fold difference of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15 or 20 or any ranges therebetween.

The levels of two or more of the nine metabolites listed above, may be used as a continuous variable using any one of a range of statistical methods that calculates the risk of an outcome in relation to multiple measurements, which can be categorical, ordinal or continuous. In such models, the metabolites or derived ratios may be used as categorical or ordinal variables using thresholds as described above. In other models, the exact value of two or more metabolites may be used in the calculation of risk. The methods employed are well known in the field, and include logistic regression, Poisson regression, distribution modelling (where differences in standard deviations between cases and controls are used to calculate likelihood ratios, which are then used in the risk calculation) and Cox proportional hazard regression. Such models include two or more of the nine metabolites listed above but may also include other variables which are also predictive of risk, such as the mother's body mass index or ratio of the serum levels of sFLTl and PIGF.

Quantifying the level of metabolite present in a sample may comprise determining the absolute concentration of the metabolite(s). In one embodiment, quantifying the level of metabolite present in a sample comprises determining the relative concentration of the metabolite compared to the concentration of a reference standard or to the total metabolite concentration of a sample. The use of relative concentrations of metabolites in diagnostic methods associated with pregnancy is commonplace in other screening contexts. Thus, in one embodiment, metabolite levels are quantified as a multiple of the median (MoM). The median may be calculated as the median concentration of the analyte in all samples or the median concentration of the analyte measured in a given period. Metabolite levels may also be log transformed and converted to standard deviations from the mean (i.e. Z-scores). The mean is typically calculated as the mean concentration of the analyte in all samples or the mean concentration of the analyte measured in a given period.

Quantification of the level of metabolites may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof. Suitable quantitative methods are readily available to the skilled person.

Biological samples that may be used according to the invention include whole blood, cells circulating in the mother's blood including those originating from the fetus or placenta, blood serum, plasma, urine, saliva, cervicovaginal fluid or other bodily fluid (stool, tear fluid, synovial fluid, sputum), or an extract or purification therefrom, or dilution thereof. Biological samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

It will be understood that methods of the invention may be performed in vitro.

In one embodiment, the biological sample is a serum sample, a dried blood sample, or is reconstituted from a dried blood sample. In one embodiment, the biological sample is serum e.g. non-fasting serum.

Typically, the level of metabolite(s) of the invention is determined by measurement of the metabolite(s) itself, or by measurement of a fragment or derivative of the metabolite(s).

Metabolite quantification may be performed using mass spectrometry (MS). Metabolite quantification may be performed by one or more method(s) selected from the group consisting of: Mass spectrometry (MS), UPLC-MS/MS, SELDI (-TOF), MALDI (-TOF), selected reaction monitoring (SRM), a 1-D gel-based analysis, a 2-D gel-based analysis, reverse phase (RP) liquid chromatography (LC), size permeation (gel filtration), ion exchange, affinity, FIPLC, UPLC, UPLC-MS/MS or other LC or LC-MS-based technique, thin-layer chromatography-based analysis or a clinical chemistry analyser. Appropriate LC MS techniques include ICAT^® (Applied Biosystems, CA, USA), or iTRAQ^® (Applied Biosystems, CA, USA). In one embodiment, liquid chromatography (e.g. high performance liquid chromatography (FIPLC) or low pressure liquid chromatography (LPLC)), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used to quantify the metabolites.

Methods of the invention may comprise analysing a sample using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) to quantify the level of metabolite(s).

In one embodiment, quantifying the level of one or more metabolite(s) of the invention comprises detecting the abundance of an ion of said one or more metabolite(s). Mass spectrometry-based detection methods suitable for use in the invention typically involve a step of derivatizing the metabolites prior to ion detection. Sample derivatization is a general term used for a chemical transformation designed to improve analytical capabilities, and it is a mainstay of analytical chemistry and instrumental analysis. Derivatizing the sample may facilitate extraction, separation and identification of metabolite(s). Thus, detection of the abundance of one or more ion(s) of metabolite(s) of the invention includes detection of the ion(s) of a derivative of metabolite(s) of the invention.

The invention provides an ion of two or more of the following metabolites: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4- cholesten-3-one and cotinine N-oxide for use in screening for risk of FSA in a subject.

The invention provides a method of screening for risk of FSA in a subject, said method comprising: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of said two or more metabolites; and (c) diagnosing the subject with an increased risk of FSA, when the level of said two or more metabolites in the biological sample is different from the reference level of said two or more metabolites.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is greater than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl-

GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is greater than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1- (l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1- (l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is greater than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC,

1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1- (l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is lower than the reference metabolite ratio.

1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1- (l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1- (l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from the subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1-

(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is greater than the reference metabolite ratio.

Metabolites of the invention may also be quantified using enzymatic assays. For example, enzymes capable of catalysing a detectable change in the metabolite(s) of the invention may be added to samples, or dilutions or extracts thereof, and products of the enzymatic reaction are quantified. For example, methods of quantifying 1,5-anhydroglucitol are known in the art and may involve adding a pyranose oxidase to a sample containing 1,5-anhydroglucitol. The oxidation of 1,5-anhydroglucitol generates hydrogen peroxide that can be detected and quantified by colorimetric methods using peroxidase. Enzymatic assays can be provided in kit form and advantageously can be designed to avoid the need for specialist equipment and may therefore be used in diverse clinical settings.

Metabolites of the invention may also be quantified using a colloidal gold aggregation method. Methods of quantifying the level of Nl,N12-diacetylspermine using colloidal gold aggregation are known in the art (Nakayama Y. et al. Oncol Lett. 2012. 3(5):970-974).

Metabolite(s) of the invention may be quantified directly or indirectly via interaction with a ligand or ligands such as an antibody or a metabolite-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the metabolite. The ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag. Quantification of metabolites may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the metabolite. In one embodiment, quantification is performed using an immunological method, optionally Enzyme-Linked Immunosorbent Assay (ELISA). In one embodiment, quantifying metabolite(s) of the invention involves detecting antibody-metabolite complexes. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the metabolites is performed using two antibodies which recognize different epitopes on a metabolite; radioimmunoassays (RIA), direct, indirect or competitive ELISA, enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay ( e.g . using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods may be performed, for example, in microtitre plate or strip format.

The invention also provides a method of screening for risk of FSA in a subject, said method comprising: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4- cholesten-3-one and cotinine N-oxide by: i) contacting the biological sample with antibodies against said two or more metabolites; and ii) detecting and/or quantifying binding between said two or more metabolites and their respective antibodies; and (c) diagnosing the subject with an increased risk of FSA, when the level of said two or more metabolites in the biological sample from the subject is different from the reference level of said two or more metabolites.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) contacting the biological sample with an antibodies against one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; and ii) detecting and/or quantifying binding between one or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) contacting the biological sample with an antibodies against one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; and ii) detecting and/or quantifying binding between one or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is greater than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) contacting the biological sample with an antibodies against one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; and ii) detecting and/or quantifying binding between one or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) contacting the biological sample with an antibodies against one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; and ii) detecting and/or quantifying binding between one or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) contacting the biological sample with an antibodies against one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; and ii) detecting and/or quantifying binding between one or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)-

2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by: i) contacting the biological sample with an antibodies against one or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; and ii) detecting and/or quantifying binding between one or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by (ii) the level of one of l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2),

Acisoga and cotinine N-oxide in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is greater than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is greater than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (d) diagnosing the subject with an increased risk of FSA, when the subject metabolite ratio is different from the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (d) diagnosing the subject with an increased risk of FGR, when the subject metabolite ratio is lower than the reference metabolite ratio.

In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; (b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by: i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine and their respective antibodies; (c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha- diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and (d) diagnosing the subject with an increased risk of LGA, when the subject metabolite ratio is greater than the reference metabolite ratio.

Immunological methods for use in the invention may be based, for example, on any of the following methods:

Immunoprecipitation, which is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen (i.e. the metabolite) present therein to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.

In particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the metabolite.

In immunonephelometry, the interaction of an antibody and target antigen on the metabolite results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer. The antigen concentration can be determined within minutes of the reaction.

Radioimmunoassay (RIA) methods employ radioactive isotopes such as 1125 to label either the antigen or antibody. The isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays.

Enzyme (EIA) immunoassays were developed as an alternative to radioimmunoassays (RIA). These methods use an enzyme to label either the antibody or target antigen. The sensitivity of EIA approaches that of RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA). ELISA methods may use two antibodies one of which is specific for the target antigen and the other of which is coupled to an enzyme, addition of the substrate for the enzyme results in production of a chemiluminescent or fluorescent signal.

Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.

In one embodiment, the immunoassay is an electrochemical luminescence (ECL) assay. In one embodiment, the ECL assay is performed using the Roche Cobas e411 immunoassay platform.

Immunological methods according to the invention can thus be performed using well-known methods. Any direct ( e.g . using a sensor chip) or indirect procedure may be used in the quantification of a metabolite of the invention. The Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention. One binding partner (hapten, antigen, ligand, aptamer, antibody, enzyme etc.) is labelled with biotin and the other partner (surface, e.g. well, bead, sensor etc.) is labelled with avidin or streptavidin. This is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one. For example a biotinylated ligand (e.g. antibody or aptamer) specific for a metabolite of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the metabolite in order to quantify a metabolite of the invention. Quantification of the immobilised antigen may then be performed by an immunological method as described herein.

The term "antibody" as used herein includes, but is not limited to: polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-ld) antibodies and epitope binding fragments of any of the above. The term "antibody" as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules for use in a method of the invention can be of any class (e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

The invention may be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices. The biosensor may incorporate an immunological method, electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect and quantify the target metabolite(s) at the anticipated concentrations found in biological samples. The metabolite(s) of the invention may be detected using a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code" or array configurations.

In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored in a thin polymer film that is sensitised to react specifically with metabolite(s). On exposure, the metabolite(s) react with the polymer leading to an alteration in the image displayed by the hologram. The test result read-out can be a change in the optical brightness, image, colour and/or position of the image. For qualitative and semi-quantitative applications, a sensor hologram can be read by eye, thus removing the need for detection equipment. A simple colour sensor can be used to read the signal when quantitative measurements are required. Opacity or colour of the sample does not interfere with operation of the sensor. The format of the sensor allows multiplexing for simultaneous detection of several substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of particular metabolite(s) of interest is feasible.

Suitably, biosensors for detection of the metabolite(s) of the invention combine biomolecular recognition with appropriate means to convert quantitation of the metabolite in the sample into a signal. Biosensors can be adapted for "alternate site" diagnostic testing, e.g. in the ward, outpatients' department, surgery, home, field and workplace.

Biosensors to detect metabolite(s) of the invention include e.g. acoustic, plasmon resonance, holographic and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the metabolite(s) of the invention.

Methods involving quantification of the metabolite(s) of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g. in the physician's office or at the patient's bedside. Suitable biosensors for performing methods of the invention include "credit" cards with optical or acoustic readers.

Methods of the invention can be performed in array format, e.g. on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format.

The invention also provides systems for analysing the level of metabolite(s) present in a sample, comparing said levels to reference level(s) and providing a diagnostic output based on whether or not there is a difference between the level of metabolite(s) in the sample and the reference level of metabolite(s).

The invention provides a computer program comprising instructions which, when the program is executed by a computer, cause the computer to compare: (i) the level of two or more metabolites in a biological sample obtained from a subject, wherein said two or more metabolites are selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; with (ii) reference level of said two or more metabolites.

In one embodiment, the computer program further comprises instructions which, when the program is executed by a computer, cause the computer to apply a mathematical model to the level of said two or more metabolites. In one embodiment, the mathematical model comprises logistic regression. In one embodiment, the mathematical model comprises distribution modelling (Royston P, Thompson SG. Model-based screening by risk with application to Down's syndrome. Stat Med.

1992 Jan 30;ll(2):257-68. PubMed PMID: 1533726). In one embodiment, the level of said metabolites corresponds to the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide.

In one embodiment, the level of said metabolites corresponds to the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one.

In one embodiment, the computer program further causes the computer to provide a diagnostic report, wherein the diagnostic report: (i) indicates a positive diagnosis of an increased risk of FSA in the subject when a difference exists between the level or combination of levels of said two or more metabolites in the biological sample and the reference level or combination of reference levels of said two or more metabolites; or (ii) indicates a negative diagnosis of an increased risk of FSA in the subject when a difference does not exist between the level or combination of levels of said two or more metabolites in the biological sample and the reference level or combination of reference levels of said two or more metabolites.

The invention also provides a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in a biological sample obtained from a subject, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in said biological sample, divided by (ii) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in said biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in said biological sample. Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one in the biological sample obtained from a subject, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in said biological sample, divided by (ii) the level of one of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in said biological sample, or the combination of the level of two or more of 1- (l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in said biological sample.

Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in a biological sample obtained from a subject, divided by (ii) the combination of the level of 5alpha-androstan- 3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in said biological sample.

Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of N1,N12- diacetylspermine in a biological sample obtained from a subject, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in said biological sample.

Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in a biological sample obtained from a subject, divided by (ii) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in said biological sample.

Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in a biological sample obtained from a subject, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl- GPC and the level of 1,5-anhydroglucitol in said biological sample.

Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the level of 1,5- anhydroglucitol in a biological sample obtained from a subject, divided by (ii) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in said biological sample.

Also provided is a computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in a biological sample obtained from a subject, divided by (ii) the level of 1,5-anhydroglucitol in said biological sample.

In one embodiment, the computer program further causes the computer to: (a) compare the subject metabolite ratio with a reference metabolite ratio; and (b) provide a diagnostic report, wherein the diagnostic report: (i) indicates a positive diagnosis of an increased risk of FSA in the subject when a difference exists between the subject metabolite ratio and a reference metabolite ratio; or (ii) indicates a negative diagnosis of an increased risk of FSA in the subject when a difference does not exist between the subject metabolite ratio and a reference metabolite ratio.

The results of any analysis, optionally in the form of a diagnostic report, will often be communicated to physicians and/or patients (or other interested parties such as researchers) in a transmittable form that can be communicated or transmitted to any of the above parties. Such a form can vary and can be tangible or intangible. The results can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. The statements and visual forms can be recorded on a tangible medium such as papers, computer readable media such as hard disks, compact disks, etc., or on an intangible medium, e.g. an electronic medium in the form of email or website on internet or intranet. In addition, results can also be recorded in a sound form and transmitted through any suitable medium, e.g. analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.

Thus, the information and data on a test result can be produced anywhere in the world and transmitted to a different location. Also described is a method for producing a transmittable form of information on levels of two or more metabolite(s) for at least one patient sample. The method comprises the steps of (1) determining levels of two or more metabolite(s) for at least one patient sample according to methods of the present invention; and (2) embodying the result of the determining step in a transmittable form. The transmittable form is the product of such a method.

Techniques for analysing levels of two or more metabolite(s) for at least one subject sample will often be implemented using hardware, software or a combination thereof in one or more computer systems or other processing systems capable of effectuating such analysis.

The computer-based analysis function can be implemented in any suitable language and/or browsers. For example, it may be implemented with C language and preferably using object-oriented high-level programming languages such as Visual Basic, SmallTalk, C++, and the like. The application can be written to suit environments such as the Microsoft Windows™ environment including Windows™ NT, Windows™ 8, Windows™ 10, and the like. In addition, the application can also be written for the Macintosh™, SUN™, UNIX or LINUX environment. In addition, the functional steps can also be implemented using a universal or platform-independent programming language. Examples of such multi-platform programming languages include, but are not limited to, hypertext markup language (HTML), JAVA™, JavaScript™, Flash programming language, common gateway interface/structured query language (CGI/SQL), practical extraction report language (PERL),

AppleScript™ and other system script languages, programming language/structured query language (PL/SQL), and the like. Java™- or JavaScript™-enabled browsers such as HotJava™, Microsoft™ Explorer™, Netscape™, Safari™ or Chrome™ can be used. When active content web pages are used, they may include Java™ applets or ActiveX™ controls or other active content technologies.

The analysis function can also be embodied in computer program products and used in the systems described above or other computer- or internet-based systems. Accordingly, described is a computer program product comprising a computer-usable medium having computer-readable program codes or instructions embodied thereon for enabling a processor to carry out FSA (or FGR or LGA more specifically) risk analysis. These computer program instructions may be loaded onto a computer or other programmable apparatus to produce a machine, such that the instructions which execute on the computer or other programmable apparatus create means for implementing the functions or steps described above. These computer program instructions may also be stored in a computer- readable memory or medium that can direct a computer or other programmable apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory or medium produce an article of manufacture including instructions which implement the analysis. The computer program instructions may also be loaded onto a computer or other programmable apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions or steps described above.

The invention provides a system for determining whether a patient has or is likely to develop FSA (or FGR or LGA more specifically). Generally speaking, the system comprises (1) computer program for receiving, storing, and/or retrieving data regarding levels of metabolites in a subject's sample and optionally clinical parameter data ( e.g ., disease-related symptoms); (2) computer program for querying this subject data; (3) computer program for concluding whether an individual has or is likely to develop FSA (or FGR or LGA more specifically) based on this patient data; and optionally (4) computer program for outputting/displaying this conclusion. In some embodiments this computer program for outputting the conclusion comprises a computer program for informing a health care professional of the conclusion.

The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products typically include computer readable media having computer- executable Instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. Basic computational biology methods are described in, for example, Setubal et al., INTRODUCTION TO COMPUTATIONAL BIOLOGY METHODS (PWS Publishing Company, Boston, 1997); Salzberg et al. (Ed.), COMPUTATIONAL METHODS IN MOLECULAR BIOLOGY, (Elsevier, Amsterdam, 1998); Rashidi & Buehler, BIOINFORMATICS BASICS: APPLICATION IN BIOLOGICAL SCIENCE AND MEDICINE (CRC Press, London, 2000); and Ouelette & Bzevanis, Attorney Docket No. 3330-01-IP Page 38 of 64 BIOINFORMATICS: A PRACTICAL GUIDE FOR ANALYSIS OF GENE AND PROTEINS (Wiley & Sons, Inc., 2nd ed., 2001); see also, U.S. Pat. No. 6,420,108.

The invention also provides a kit comprising (i) reagents and/or a biosensor capable of quantifying two or more metabolites selected from the list consisting of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; and (ii) instructions for use in screening for risk of FSA.

Suitably a kit may contain one or more components selected from the group: a ligand specific for the metabolite or a structural/shape mimic of the metabolite, one or more controls, one or more reagents and one or more consumables; optionally together with instructions for use of the kit in accordance with any of the methods defined herein. Kits may additionally contain a biosensor capable of quantifying a metabolite.

The identification of metabolite(s) for FSA (or FGR or LGA more specifically) permits integration of diagnostic procedures and therapeutic regimes. Combinations of two or more metabolites of the invention can be used to screen subjects who have FSA (or FGR or LGA more specifically) or who are at risk of developing FSA (or FGR or LGA more specifically). Screening can be used to identify subjects who require further monitoring and who may require intervention, e.g. early delivery.

The invention will now be described by way of the following non-limiting examples.

EXAMPLES

Methods

The approach of the study was as follows: (i) identify candidate metabolite predictors using the 12, 20 & 28wkGA Pregnancy Outcome Prediction (POP) study samples, and (ii) validate predictors using the 36wkGA POP study sample and identify those which were independently predictive of term FGR and (iii) test whether identified metabolite markers are predictive of LGA.

Study design

The POP study has been described previously in detail. It was a prospective cohort study of unselected nulliparous women with a singleton pregnancy attending the Rosie Hospital, Cambridge, UK, between Jan 2008 and Jul 2012. Participants had blood sampling and fetal biometry at 12, 20, 28 and 36wkGA. In total, 4212 women completed the POP study. After the exclusion of miscarriages, fetal deaths prior to 23wkGA and terminations (total n=29) and women who did not have any blood samples available (n=6), 4177 women remained in the cohort. A random sub-cohort (n=325) was selected from this population for use in the present study. Outcome data were obtained by linkage to the hospital's electronic database on deliveries. Ethical approval was obtained from the Cambridgeshire 2 Research Ethics Committee (reference number 07/H0308/163). All study participants gave written informed consent. This study is reported according to the STARD 2015 guidelines for reporting diagnostic accuracy studies (http://www.stard-statement.org/).

A case-cohort design within the POP study was used for the metabolomics analysis (4). FGR at term was defined as delivery at >37wkGA and (a) customized(5) birth weight (BW) centile <10^th combined with abdominal circumference growth velocity (ACGV) in the lowest decile between 20 and 36 wkGA or (b) customized birth weight (BW) centile <3^rd. LGA was defined as birthweight >90^th centile, >95^th centile or >4kg. A random sample of the cohort was selected as a comparison group.

Biochemical analyses

Measurement of protein levels was performed on maternal serum using the Roche Cobas e411 immunoassay platform, as previously described(6). Metabolomic analysis was performed by Metabolon (Research Triangle Park, NC, USA), blinded to the patients' clinical information and pregnancy outcome, as previously described(7). Ultrahigh Performance Liquid Chromatography- Tandem Mass Spectrometry (UPLC-MS/MS) was used as the analysis platform(8). Metabolite concentrations were quantified using area-under-the-curve of primary MS ions and were expressed as the multiple of the median value for all batches processed on the given day. 1193 untargeted metabolites were measured from each sample, 837 of known structural identity. Metabolomics was performed on non-fasting serum obtained at ~12wkGA, ~20wkGA, ~28wkGA and 36wkGA.

Statistical analysis

Scaled imputed metabolite values (multiples of the median) were log-transformed and converted to z scores. Initial selection of predictive metabolites involved fitting longitudinal linear mixed models for each metabolite using measurements from 12wkGA, 20wkGA, and 28wkGA, to generate a difference in the metabolite means and associated P value in the maternal serum at 20wkGA and/or

28wkGA (composite Chi-squared test) comparing term FGR cases and controls. The inventors included interaction terms between term FGR and gestational age to identify differences and the metabolites were then ranked by the composite P value at 20/28wkGA. Excess of low P values was tested using a one-sample Kolmogorov-Smirnov test against the theoretical uniform distribution of P values between 0 and 1. To validate findings, the 36wkGA sample from the same women in the FGR cases and controls groups were compared using linear regression with a Bonferroni corrected P value to minimize false positive results. Forward-stepwise logistic regression (P<0.05 for entry and P<0.1 for removal) was used to select independent predictors of term FGR. Standard screening statistics were calculated from 2x2 tables in the POP study cohort, weighting the comparison group by the inverse of the sampling fraction. Prediction of term FGR was further assessed using the area under the receiver operating characteristic (ROC) curve (AUC). An AUC value of 0.5 indicates a non-informative test, and higher levels of AUC indicate progressively better prediction up to a maximum of 1.0 which indicates perfect prediction.

Table 1. Characteristics of the Pregnancy Outcome Prediction study cohort in the metabolomics analysis of fetal growth restriction

FGR term Term controls without FGR

Characteristic

(N=175) (N=299)

Maternal characteristics

Age, years 30 (26 to 34) 30 (27 to 33)

Age stopped FTE >21 years 82 (47%) 165 (55%) Missing 4 (2%) 1 (<1%)

Height, cm 166 (161 to 169) 165 (161 to 169) BMI, kg/m² 24 (22 to 28) 24 (22 to 28) Smoker 29 (17%) 12 (4%)

Any alcohol consumption 9 (5%) 10 (3%) Deprivation, score 9.76 (5.34 to 15.07) 8.53 (5.94 to 14.17) Deprivation, rank 24142 25734

(18144 to 29575) (19045 to 28876)

Deprivation rank quintile

1 (most deprived) 0 (0%) 0 (0%)

2 19 (11%) 18 (6%)

3 31 (18%) 59 (20%)

4 47 (27%) 72 (24%)

5 (least deprived) 68 (39%) 139 (46%) Missing 10 (6%) 11 (4%) White ethnicity 166 (95%) 280 (94%)

Missing 2 (1%) 6 (2%)

Married 107 (61%) 218 (73%)

Diabetes

Type 1 or type 2 DM 1 (1%) 0 (0%)

Gestational DM 5 (3%) 11 (4%)

Essential HT 7 (4%) 9 (3%)

Pre-existing renal disease 3 (2%) 2 (1%)

Preeclampsia 15 (9%) 17 (6%)

Birth outcomes

Birth weight, g 2680 (2410 to 2860) 3495 (3170 to 3800) Gestational age, weeks 40.1 (39.1 to 41.1) 40.3 (39.4 to 41.3) Female fetal sex 91 (52%) 156 (52%) Induction of labour 61 (35%) 108 (36%) Mode of delivery

Spontaneous vaginal 96 (55%) 147 (49%) Assisted vaginal 33 (19%) 63 (21%) Intrapartum caesarean 21 (12%) 59 (20%) Pre-labour caesarean 25 (14%) 28 (9%) Missing 0 (0%) 2 (1%)

Data are expressed as median (interquartile range (IQR)) or n (%) as appropriate. For fields where there is no category labelled "missing", data were 100% complete. Maternal age was defined as age at recruitment. All other maternal characteristics were defined by self-report at the 20wkGA questionnaire, from examination of the clinical case record, or linkage to the hospital's electronic databases. The weight measurement used in the BMI calculation was made at the 12wkGA visit. Socio-economic status was quantified using the Index of Multiple Deprivation 2007, which is based on census data from the area of the mother's postcode (9). Deprivation score is the combined sum of the weighted, exponentially transformed domain rank of the domain score, and higher values indicate more deprivation. Conversely, the most deprived area has the lowest rank (=1) and the least deprived area has the highest rank (=32482). A national reference distribution from 2010 has been used to analyse the rank in quintiles (l=most deprived, 5=least deprived). Preeclampsia was defined on the basis of the 2013 ACOG criteria. Abbreviations: FTE, full time education; BMI, body mass index; DM, diabetes mellitus; PE, preeclampsia; FIT, hypertension.

Results

The characteristics of the POP study groups are summarized in (Table 1). Women who delivered a baby with term FGR were more likely to be smokers. In FGR cases, the median birth weight was ~23% lower, and there were higher rates of preeclampsia and pre-labour caesarean delivery but lower rates of intrapartum caesarean.

After excluding eight xenobiotic metabolites with minimal variation, 829 identified metabolites were analysed. Longitudinal linear mixed effects regression of the 12, 20 and 28wkGA samples generated a composite P value for each metabolite and the P value distribution was skewed towards lower values (Kolmogorov-Smirnov test P=0.002, Figure 1). The 100 metabolites with the lowest P values were selected for further study. Validation was accepted if the P value at 36wkGA was below the Bonferroni-corrected threshold P<5xl0-4, yielding a list of 22 metabolites which were validated using this highly conservative approach (Table 2). Metabolites which met the highly conservative significance criteria and thus found to be strongly associated with FGR included l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), acisoga, estriol 3 sulfate, 4- cholesten-3-one and cotinine-n-oxide.

A correlation matrix demonstrated that some of these metabolites were strongly correlated with each other and with maternal characteristics and the sFLTl:PIGF ratio (Table 3). As the aim of the study was to generate novel, independent predictors, the inventors used a forward stepwise logistic regression model which included the sFLTl:PIGF ratio, maternal age (linear and quadratic terms) and BMI as well as the 22 validated metabolites. Nine of the 22 metabolites were independently predictive of FGR using this approach and five of these improved the prediction over the sFltl:PIGF ratio.

The unadjusted and adjusted odds ratios (95% Cl) of five of the metabolites of the invention at 36wkGA in relation to term FGR are shown in Table 4. Two metabolites which were negatively associated with term FGR (5alpha-androstan-3alpha,17alpha-diol disulfate and N1,N12- diacetylspermine) had an increasing trend throughout pregnancy (Figure 2A and 2B), whereas the two metabolites which were positively associated with the risk of term FGR at 36wkGA (l-(l-enyl- stearoyl)-2-oleoyl-GPC (P-18:0/18:l) and 1,5-anhydroglucitol) had a declining trend throughout pregnancy (Figure 2C and 2D). Flence, each of the four associations observed with FGR represented attenuation of the physiological change observed in normal pregnancies. Table 2. Known metabolites with different maternal serum levels by term FGR, included into top 100 metabolites by P value at 20/28 weeks of gestational age (wkGA), validated using a 36wkGA measurement at P<5xlQ ⁰⁴.

Metabolic P value Direction

P value

Metabolite Pathway 20-28 36wkGA of wkGA Association

5alpha-androstan-3alpha,17alpha-diol Steroid

5x10 ⁷ 3xl0 disulfate ^u

Estriol 3-sulfate Steroid 3x10 ⁶ 9x10 ⁸

4-cholesten-3-one Sterol 0.004 2xl0 ⁷ l-(l-enyl-stearoyl)-2-oleoyl-GPC (P- Plasmalogen

0.011 2x10

18:0/18:1) ^{7 +}

Pregnanolone/allopregnanolone sulfate Steroid 0.003 3x10 ⁷

Glycolysis, Gluconeogenesis, +

1,5-anhydroglucitol (1,5-AG) 8xl0 and Pyruvate Metabolism ⁴ 8xl0^~7

5alpha-pregnan-3alpha,20beta-diol Steroid

0.065 2x1.0 disulfate 1 ⁶

Cotinine N-oxide Tobacco Metabolite 9xl0 ⁷ 3xl£f⁶ -s- 4-androsten-3beta,17beta-diol Steroid +

0.026 4xl0 monosulfate (2) ^~6

4-androsten-3beta,17beta-diol Steroid

0.023 7xlG ⁶ monosulfate (1) l-(l-enyl-palmitoyl)-2-palmitoleoyl-GPC Plasmalogen

0.003 8x10

(P-16:0/16:l)* ⁶

Hydroxycotinine Tobacco Metabolite 8xl0 ⁶ lxlO ⁵ +

Nl,N12-diacetylspermine Polyamine Metabolism 0.048 lxlO ⁵

Acisoga Polyamine Metabolism 0.004 4xl0 ⁵ +

17alpha-hydroxypregnanolone Steroid

6x10 ⁴ 4xl0 glucuronide ⁵

5alpha-pregnan-3beta,20beta-diol Steroid

0.019 5xl0 ⁵ monosulfate (1)

3-hydroxycotinine glucuronide Steroid 2x10 ⁵ lxlO ⁴ +

Phenylalanine and Tyrosine +

O-cresol sulfate 2x10

Metabolism ⁴

Progesterone Steroid 0.004 4xl0 ⁴

Pregnanediol-3-glucuronide Steroid 0.005 4xl0 ⁴

Metabolites (n=20) are sorted by P value at 36wkGA. Differences at 20wkGA or 28wkGA (composite hypothesis) between FGR cases and control cases were tested using a Chi-squared test (testparm postestimation command in Stata). Validation at 36wkGA was performed using a t test, indicates compounds that have not been officially confirmed based on a standard. Nine metabolites selected for further assessment following a stepwise procedure are marked in bold.

Each of the five metabolites were able to discriminate between FGR cases and controls with AUCs of between 0.64 and 0.69 (Table 4). The inventors also assessed the predictive ability of the metabolites in combination. The product of the two metabolites positively associated with the risk of FGR (l-(l-enyl-stearoyl)-2-oleoyl-GPC (P-18:0/18:l) and 1,5-anhydroglucitol) produced an AUC of

0.70 and the product of the two metabolites negatively associated with the risk of FGR (5alpha- androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine) produced an AUC of 0.71 (Table 4). The inventors also calculated the ratio of the product of the two positively associated metabolites divided by the product of the two negatively associated metabolites (referred to herein as the metabolite ratio). This approach is advantageous as it avoids the need for complex multivariate statistical models while providing a similar level of diagnostic accuracy (multivariate statistical model AUC=0.783; metabolite ratio AUC=0.778; P= 0.59). The AUC for the metabolite ratio for term FGR at 36wkGA was notably higher than for the sFLTl:PIGF ratio (metabolite ratio AUC = 0.778; sFLTl:PIGF ratio AUC = 0.636, Figure 3). Beneficially, the metabolite ratio at 28 wkGA produced an AUC of 0.72 (95% Cl: 0.67 to 0.77) indicating that the invention is advantageously suitable for early detection of FGR (Figure 4).

Within the Examples, unless specified otherwise, the "metabolite ratio" refers to: (l-(l-enyl- stearoyl)-2-oleoyl-GPC x 1,5-anhydroglucitol) / (5a-androstan-3ot,17ot-diol disulfate x N1,N12- diacetylspermine).

In some circumstances, 5a-androstan-3ot,17ot-diol disulfate can be difficult to measure. The inventors determined that estriol 3-sulfate (which is easier to measure) is strongly correlated with 5a-androstan-3ot,17ot-diol disulfate (Table 3), and therefore tested whether estriol 3-sulfate can provide a suitable experimental surrogate for 5a-androstan-3ot,17ot-diol disulfate. Advantageously, ratios calculated using estriol 3-sulfate as a "surrogate" for 5a-androstan-3ot,17ot-diol disulfate achieved similarly high predictive performance as ratios incorporating 5a-androstan-3ot,17ot-diol disulfate, including when combined with ultrasound (see Surrogate metabolite ratio data in Tables 4 and 5).

Table 4. Metabolite measurements and their products and ratios at 36wkGA in relation to FGR^a at term. Odds ratios (95% confidence intervals) of the unadjusted and adjusted metabolite measurements at 36 wkGA in relation to term FGR (n=175) in the case cohort sample (n=299 term controls without FGR) and area under the ROC curve.

Odds ratio^d (95% Cl)

Metabolite AUC^b (95% Cl) P^c Unadjusted Adjusted⁶ Fully adjusted^f

(A) l-(l-enyl-stearoyl)-2-oleoyl-GPC (P-18:0/18:l) 0.64 (0.58 to 0.69) 2.3xlCT 1.76 (1.41 to 2.21) 1.68 (1.34 to 2.12) 1.76 (1.38 to 2.23)

(B) 1,5-anhydroglucitol (1,5-AG) 0.65 (0.60 to 0.71) 8.4xl0^-7 1.79 (1.40 to 2.27) 1.65 (1.29 to 2.11) 1.62 (1.26 to 2.07)

(C) 5a-androstan-3a,17a-diol disulfate 0.69 (0.64 to 0.74) 3.2x10¹¹ 0.51 (0.41 to 0.64) 0.52 (0.42 to 0.65) 0.51 (0.41 to 0.64)

(D) Nl,N12-diacetylspermine 0.66 (0.61 to 0.71) l.lxlO^-5 0.63 (0.51 to 0.79) 0.58 (0.45 to 0.74) 0.58 (0.45 to 0.74) Numerator of the metabolite ratio (A x B) 0.70 (0.64 to 0.75) 7.9x10¹¹ 2.18 (1.70 to 2.81) 2.02 (1.56 to 2.61) 2.01 (1.55 to 2.61) Denominator of the metabolite ratio (C x D) 0.71 (0.66 to 0.76) 7.6x10¹² 0.49 (0.39 to 0.62) 0.48 (0.38 to 0.60) 0.47 (0.37 to 0.60)

(E) Estriol 3-sulfate 0.65 (0.59 to 0.70) 8.6xl0^-8 0.58 (0.47 to 0.71) 0.55 (0.44 to 0.68) 0.51 (0.41 to 0.65)

Surrogate metabolite ratio (A x B) / (E x D) 0.77 (0.72 to 0.81) 2.9xl0^-20 2.82 (2.18 to 3.64) 2.88 (2.21 to 3.76) 2.96 (2.25 to 3.89) Metabolite ratio (A x B) / (C x D) 0.78 (0.73 to 0.82) l.lxlO^-21 2.93 (2.25 to 3.80) 2.86 (2.20 to 3.73) 2.82 (2.17 to 3.68)

The total number of women who had metabolite measurements at 36 wkGA was 437, including 162 cases of FGR and 275 controls born at term. FGR at term was defined as delivery at >37wkGA with customised birth weight <3^rd percentile, or customised birth weight <10^th percentile with abdominal circumference growth velocity in the lowest decile (see Methods). ^bAUC was based on the metabolites alone. Calculated from linear regression using Wald test, with the null hypothesis that the coefficient = 0. ^dOdds ratios were given for one standard deviation higher value of the log-transformed metabolite, product or ratio. ^eAdjusted for the log-transformed sFlt-1: PIGF ratio at 36wkGA. Additionally adjusted for maternal age (linear and quadratic term) and body mass index at 12wkGA. wkGA, weeks of gestational age; FGR, fetal growth restriction; Cl, confidence interval; AUC, area under the ROC curve.

Table 5. Diagnostic effectiveness of ultrasonic and biochemical screening at 36wkGA for delivery of an infant with FGR^a at term.

Screening test TP/FP TN/FN Screen† Positive LR Negative LR Sensitivity Specificity PPV^C NPV^C DOR

Comp (95% Cl) (95% Cl) (95% Cl) (95% Cl) (95% Cl) (95% Cl) (95% Cl)

Ultrasonic EFW <10^th 110/39 234/50 17.1 4.8 0.36 68.8 85.7 18.0 98.4 13.2

(3.5-6.6) (0.29-0.46) (61.1-75.5) (81.0-89.4) (13.2-24.1) (97.8-98.8) (8.2-21.0) sFLTl:PIGF ratio >38 49/33 240/111 12.5 2.5 0.79 30.6 87.9 10.4 96.5 3.2

(1.7-3.8) (0.71-0.88) (23.9-38.3) (83.5-91.3) (6.9-15.4) (95.7-97.2) (1.9-5.3) bMetabolite ratio >85^th 86/37 236/74 15.0 4.0 0.54 53.8 86.4 15.3 97.6 7.4

(2.8-5.5) (0.45-0.64) (45.9-61.4) (81.8-90.0) (10.9-21.1) (96.9-98.2) (4.7-11.9)

Ultrasonic EFW <10^th 34/6 267/126 3.1 9.7 0.81 21.3 97.8 30.6 96.5 12.0 and sFLTl:PIGF ratio >38 (4.2-22.5) (0.74-0.87) (15.5-28.4) (95.2-99.0) (15.1-52.2) (95.7-97.1) (4.9-27.4)

Ultrasonic EFW <10^th 56/4 269/104 3.1 23.9 0.66 35.0 98.5 52.1 97.1 36.2 and metabolite ratio >85^th (8.8-64.6) (0.59-0.74) (27.9-42.8) (96.1-99.5) (27.7-75.6) (96.4-97.7) (13.7-94.9)

Surrogate ratio >85^th 80/37 236/80 14.7 3.7 0.58 50.0 86.4 14.4 97.4 6.4

(2.6-5.2) (0.49-0.68) (42.2-57.8) (81.8-90.0) (10.2-20.0) (96.7-98.0) (4.0-10.2)

Ultrasonic EFW <10^th 54/5 268/106 3.8 18.4 0.67 33.8 98.2 45.7 97.0 27.3 and surrogate ratio >85^th (7.5-45.1) (0.60-0.75) (26.8-41.5) (95.7-99.2) (24.6-68.4) (96.3-97.6) (10.9-64.4)

The total number of women in this analysis was 433, including 160 cases of FGR and 273 controls, due to missing values in EFW for two cases and two controls. ^aFGR at term was defined as delivery at >37wkGA with customised birth weight <3^rd percentile, or customised birth weight <10^th percentile with abdominal circumference growth velocity in the lowest decile (see Methods). ^bMetabolite ratio is the ratio of two products of metabolites (see Methods). As the sFLTl:PIGF ratio >38 approximates to the 85^th percentile in the whole POP study cohort, we selected the same threshold in this analysis. ^cDue to the case-cohort design, the proportion of screen positives was calculated in the random subcohort, i.e. comparator group, in women who had all three measurements (EFW, sFLTl:PIGF, metabolite ratio) available (n=287 including 14 cases of FGR and 273 non-cases), and PPV and NPV were weighted by the inverse of the random subcohort sampling fraction. The proportion of screen positives, sensitivity, specificity, PPV and NPV are given in percentages (%). wkGA, weeks of gestational age; FGR, fetal growth restriction; TP, true positive; FP, false positive; TN, true negative, FN, false negative; Screen†, screen positive; Comp, comparator group; LR, likelihood ratio; Cl, confidence interval; PPV, positive predictive value; NPV, negative predictive value; DOR, diagnostic odds ratio; EFW, estimated fetal weight; sFLTl, soluble fms-like tyrosine kinase 1; PIGF, placenta growth factor.

Surrogate ratio: Estriol 3-sulfate used instead of 5a-androstan-3ot,17ot-diol disulfate in the metabolite ratio.

The inventors further compared the diagnostic effectiveness of the metabolite ratio in combination with ultrasound. Diagnostic effectiveness was studied using various combinations and threshold values of the metabolite ratio, estimated fetal weight (EFW) and the sFLTl:PIGF ratio measured at 36wkGA, in relation to FGR at term in the POP study (Table 5). Generally, the combination of EFW and the metabolite ratio was the most effective, and the addition of the sFLTl:PIGF ratio did not improve diagnostic effectiveness. The combination of EFW<10^th and metabolite ratio >85^th percentile identified ~35% of the term FGR cases while giving a false positive rate of ~2%. The positive likelihood ratio (LR+) was ~23.9 and a half of women who tested positive experienced the outcome. The association between the ratio and term FGR was generally similar across different strata of maternal characteristics (Figure 5). Whereas sFLTl:PIGF was more strongly associated with term FGR with preeclampsia, the metabolite ratio was equally strongly associated with FGR regardless of preeclampsia status (Figure 6).

The inventors have identified nine metabolites which are independently predictive of FGR. Combinations of these markers are highly advantageous in diagnosing an increased risk of FGR. For example, the inventors have found that a ratio of four metabolites provided clinically useful prediction of the risk of FGR at term. This exemplary ratio was calculated by the product of two metabolites positively associated with term FGR divided by the product of two metabolites negatively associated with term FGR. The AUC indicated that the ratio was approximately twice as predictive as the angiogenic biomarker ratio, sFLTl:PIGF. Similarly, when both ratios were set to a similar false positive rate, the metabolite ratio had a positive likelihood ratio of 4.5 whereas sFLTl:PIGF had a positive likelihood ratio of 2.5. The combination of being in the lowest quintile of ultrasonic EFW and the highest quintile of the metabolite ratio had a positive likelihood ratio for term FGR of >20 and detected the majority of cases for a 3% false positive rate (i.e. specificity of 98.5%). Combinations of metabolites of the invention (including the novel ratio described herein) provide a clinically desirable test for term FGR. As the metabolites identified herein are independently predictive of FGR, advantageous diagnostic results may be expected using any combination thereof.

Interestingly, the direction of the association with FGR was the opposite of the direction of the association with advancing gestational age, i.e. if the metabolite increased with advancing pregnancy, low levels were associated with FGR and vice versa. This observation suggests that FGR is associated with attenuation of the physiological metabolic changes associated with normal pregnancy. Without wishing to be bound by theory, the inventors believe that the most likely explanation is dysregulation of normal placental metabolism, because placental dysfunction is thought to underlie many cases of FGR.

Flaving identified nine metabolites which are independently predictive of FGR, the inventors tested whether any of these metabolites would also be predictive of LGA. The ability to detect both types of FSA using the same metabolites is highly desirable because it reduces the minimum number of metabolites required to be tested, which in turn simplifies methods of detection, allows for use of less complicated detection apparatus, and reduces time and cost.

As shown in Table 7, metabolites which are independently predictive of FGR unexpectedly achieved strong prediction of LGA, either alone or in combination. Moreover, prediction of LGA was achieved when using different definitions thereof, including birthweight >90^th centile, 95^th centile and >4kg.

The inventors further tested the ability of metabolite ratios to identify an increased risk of LGA.

Surprisingly, the metabolite ratio ((l-(l-enyl-stearoyl)-2-oleoyl-GPC x 1,5-anhydroglucitol) / (5a- androstan-3a,17a-diol disulfate x Nl,N12-diacetylspermine)) was predictive across the full range of birthweights, i.e. it was predictive of both FGR and LGA (Figure 7). As shown in Table 8, the metabolite ratio achieved unexpectedly robust detection of LGA which was comparable to detection of FGR. Both the numerator of the metabolite ratio (l-(l-enyl-stearoyl)-2-oleoyl-GPC x 1,5- anhydroglucitol) and the denominator (5a-androstan-3ot,17ot-diol disulfate x N1,N12- diacetylspermine) were predictive of FGR (Table 4) and LGA (Tables 6 and 7) demonstrating that the use of two or more metabolites of the invention can be used to predict FSA (or more specifically,

FGR or LGA).

Thus, the metabolites of the invention successfully achieve robust detection of FSA, including FGR and LGA more specifically.

Table 6

Exposure_ b (95% Cl) P value

Numerator of the metabolite ratio (A x B) -0.23 (-0.35 to -0.11) lxlO^-4

Denominator of the metabolite ratio (C x D) 0.27 (0.17 to 0.38) lxlO^-6

Metabolite ratio (A x B) / (C x D) -0.37 (-0.47 to -0.26) 7xl0 ⁿ

Regression coefficients (b) are given for one standard deviation increase in the log-transformed metabolite product or ratio. Customized birthweight percentiles were obtained using the Gestation- Related Optimal Weight bulk calculator (GROW, version 6.7.8.1, Perinatal Institute, Birmingham, UK), and these were turned into z scores using the invnormal function in Stata. Cases of preeclampsia and gestational diabetes were excluded from the analysis. (A) l-(l-enyl-stearoyl)-2- oleoyl-GPC (P-18:0/18:l); (B) 1,5-anhydroglucitol (1,5-AG); (C) 5alpha-androstan-3alpha,17alpha-diol disulfate; (D) Nl,N12-diacetylspermine.

Table 7

BW>90^th BW>95^th BW>4000g

Metabolite AUC (95% Cl) P AUC (95% Cl) P AUC (95% Cl) P

(A) l-(l-enyl-stearoyl)-2-oleoyl-GPC (P-18:0/18:l) 0.80 (0.70 to 0.89) 2.4xl0⁵ 0.77 (0.64 to 0.89) 0.032 0.75 (0.66 to 0.83) 8.0xl0^-6

(B) 1,5-anhydroglucitol (1,5-AG) 0.74 (0.63 to 0.85) 3.0xl0⁵ 0.85 (0.77 to 0.92) 1.8xl0⁴ 0.67 (0.57 to 0.77) 3.6xl0^-4

(C) 5a-androstan-3oc,17oc-diol disulfate 0.59 (0.45 to 0.72) 0.37 0.66 (0.40 to 0.91) 0.18 0.52 (0.41 to 0.63) 0.59

(D) Nl,N12-diacetylspermine 0.67 (0.55 to 0.80) 0.016 0.62 (0.43 to 0.80) 0.24 0.57 (0.47 to 0.66) 0.11

(E) 4-androsten-3beta,17beta-diol monosulfate (2) 0.66 (0.57 to 0.75) 0.022 0.63 (0.50 to 0.76) 0.29 0.66 (0.58 to 0.73) 0.0030

(F) Acisoga 0.51 (0.39 to 0.63) >0.99 0.54 (0.33 to 0.75) 0.63 0.55 (0.47 to 0.63) 0.49

(G) Estriol 3-sulfate 0.62 (0.50 to 0.74) 0.11 0.62 (0.41 to 0.84) 0.32 0.55 (0.44 to 0.66) 0.38

(H) 4-cholesten-3-one 0.64 (0.52 to 0.76) 0.035 0.72 (0.51 to 0.92) 0.062 0.56 (0.46 to 0.67) 0.22

Numerator of the metabolite ratio (A x B) 0.82 (0.73 to 0.91) 2.9xl0⁸ 0.88 (0.81 to 0.95) 2.9xl0⁵ 0.76 (0.67 to 0.84) 2.8xl0^-7

Denominator of the metabolite ratio (C x D) 0.66 (0.52 to 0.79) 0.045 0.68 (0.42 to 0.93) 0.12 0.57 (0.46 to 0.67) 0.19

Metabolite ratio developed for FGR (A x B) / (C x D) 0.83 (0.74 to 0.93) 4.5xl0⁷ 0.88 (0.75 to 1.00) 1.2xl0⁴ 0.71 (0.61 to 0.80) 3.0xl0^-5

Metabolite ratio with (I) cotinine N-oxide 0.84 (0.74 to 0.93) 5.0xl0⁷ 0.88 (0.76 to 1.00) 1.5xl0⁴ 0.71 (0.62 to 0.81) 1.8xl0^-5 (A x B x I) / (C x D)

Surrogate ratio developed for FGR (A x B) / (G x D) 0.85 (0.77 to 0.94) 5.3xl0⁹ 0.87 (0.75 to 0.99) 9.7xl0⁵ 0.72 (0.63 to 0.81) 2.7xl0^-6

Surrogate ratio excluding plasmalogen: B / (G x D) 0.79 (0.68 to 0.89) 4.1c10^-6 0.83 (0.67 to 1.00) 9.2xl0⁴ 0.66 (0.56 to 0.75) 6.9xl0^-4 Steroid ratio 0.67 (0.57 to 0.77) 0.016 0.69 (0.53 to 0.84) 0.092 0.64 (0.55 to 0.73) 0.0067 Polyamine ratio 0.60 (0.46 to 0.74) 0.10 0.54 (0.30 to 0.79) 0.70 0.58 (0.48 to 0.68) 0.095

Steroid / polyamine ratio 0.67 (0.54 to 0.80) 0.0051 0.64 (0.40 to 0.89) 0.14 0.64 (0.55 to 0.74) 0.0023

The metabolite ratio [(A x B) / (C x D)] developed to predict fetal growth restriction was calculated from four metabolites measured at 36wkGA: (A) 1-(1- enyl-stearoyl)-2-oleoyl-GPC (P-18:0/18:l); (B) 1,5-anhydroglucitol (1,5-AG); (C) 5alpha-androstan-3alpha,17alpha-diol disulfate; (D) N1,N12- diacetylspermine. The steroid ratio was calculated from two metabolites: 4-androsten-3beta,17beta-diol monosulfate (2) / 5alpha-androstan- 3alpha,17alpha-diol disulfate. Polyamine ratio was calculated from two metabolites: Nl,N12-diacetylspermine / Acisoga. Macrosomia was not included as cases in the case-cohort selection. This analysis was restricted to women from the comparator group who delivered at term and had metabolite data from the 36wkGA visit (n=289). Customized birthweight percentiles (GROW bulk calculator, version 6.7.8.1, Perinatal Institute, Birmingham, UK) were used to define macrosomia using 95^th and 90^th centile cut-offs. Additionally, macrosomia was defined using 4000g cut-off of birthweight. AUC was based on the metabolite ratio alone. The P value (2-sided) was calculated from linear regression using Wald test, with the null hypothesis that the coefficient = 0. Cl, confidence interval; AUC, area under the ROC curve; wkGA, weeks of gestational age; GROW, Gestation-Related Optimal Weight.

Table 8 Metabolite ratio, LGA and macrosomia at term.

Unadjusted Adjusted

Term outcome n(cases) AUC (95% Cl) P OR (95%CI) OR (95%CI)

LGA>95^th 8 0.88 (0.75 to 1.00) 1.2xl0⁴ 0.19 (0.08 to 0.46) 0.16 (0.06 to 0.43)

LGA>90^th 21 0.83 (0.74 to 0.93) 4.5xl0⁷ 0.24 (0.14 to 0.43) 0.22 (0.12 to 0.41)

LGA>80^th 50 0.69 (0.61 to 0.77) 6.1xl0⁵ 0.48 (0.34 to 0.69) 0.47 (0.32 to 0.68)

BW>4000g 37 0.71 (0.61 to 0.80) 3.0xl0⁵ 0.42 (0.28 to 0.63) 0.40 (0.26 to 0.61)

BW>4500g 6 0.77 (0.60 to 0.94) 2.1xl0² 0.34 (0.14 to 0.83) 0.29 (0.11 to 0.80)

The metabolite ratio [(A x B) / (C x D)] was calculated from four metabolites measured at 36wkGA: (A) l-(l-enyl-stearoyl)-2-oleoyl-GPC (P-18:0/18:l); (B) 1,5-anhydroglucitol (1,5-AG); (C) 5alpha- androstan-3alpha,17alpha-diol disulfate; (D) Nl,N12-diacetylspermine. LGA and macrosomia were not included as cases in the case-cohort selection. This analysis was restricted to women from the comparator group who delivered at term and had metabolite data from the 36wkGA visit (n=289). Customized birthweight percentiles (GROW bulk calculator, version 6.7.8.1, Perinatal Institute, Birmingham, UK) were used to define LGA using 95^th, 90^th and 80^th centile cut-offs. Macrosomia was defined using 4000g and 4500g cut-offs of birth weight. AUC was based on the metabolite ratio alone. The P value (2-sided) was calculated from linear regression using Wald test, with the null hypothesis that the coefficient = 0. ORs were given for one standard deviation higher value of the log-transformed metabolite ratio. The OR was reported as 1) unadjusted and 2) adjusted for the log- transformed sFlt-l:PIGF ratio at 36wkGA, maternal age (linear and quadratic term) and body mass index at 12wkGA. OR, odds ratio; Cl, confidence interval; AUC, area under the ROC curve; LGA, large for gestational age; BW, birth weight; wkGA, weeks of gestational age; GROW, Gestation-Related Optimal Weight.

One of the major challenges with "omic" analyses is the potential for false discovery. The statistical basis for this is multiple testing. Specifically, many studies using "omic" methods evaluate large number of candidate predictors in a relatively small number of cases. The large number of hypothesis tests means that many apparently statistically significant associations are false discoveries due to type 1 statistical error. In the present study the inventors took multiple precautions to address the problem of false discovery. First, the initial selection of candidates for FGR diagnosis assessed the level of each metabolite across three serial measurements, performed at

12, 20 & 28wkGA. Metabolites were selected on the basis of a trend of change across the three time points, which the inventors believed would enrich the list of candidates with true associations. In contrast, associations explained by random fluctuation are less likely to be consistently observed across serial measurements. Second, the inventors used the 36wkGA sample for internal validation. Moreover, when the inventors assessed associations using analysis of this sample, the inventors employed the highly conservative Bonferroni method for selection, with a P value of <5x10-4. All four of the metabolites used in the exemplary ratio had a reduction in P value from the 20/28wkGA test to the 36wkGA validation of three or more orders of magnitude. The P values associated with the metabolites of the invention makes it extremely unlikely that they were false discoveries.

The inventors used Bonferroni correction of P values to ensure that only clinically useful predictors of FGR were identified. Diagnostic effectiveness generally requires large effect sizes. While this approach may have rejected associations with FGR which were true, it advantageously allowed the identification of robust associations which were diagnostically effective and robust.

In conclusion, the inventors employed metabolomics on serial maternal serum samples from a large number of first pregnancies and successfully identified nine metabolites which are independently predictive of FGR. Advantageously, the nine identified metabolites were also predictive of an increased risk of LGA. The metabolites of the invention offer considerable diagnostic advantages, which are enhanced when two or more of these metabolites are considered in combination e.g. via determination of a metabolite ratio. Methods of the invention may be used alone, or in combination with existing screening techniques for FSA, such as ultrasound. The diagnostic effectiveness of the invention was such that a randomised controlled trial of screening and intervention can reasonably be expected to demonstrate clinical effectiveness. References

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Claims

1. A method of screening for risk of fetal size abnormality (FSA) in a subject, the method comprising:

(a) quantifying in a biological sample obtained from the subject the level of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; and

(b) comparing the level of said two or more metabolites in the biological sample with reference level of said two or more metabolites; wherein a difference between the level of said two or more metabolites in the biological sample and the reference level of said two or more metabolites is indicative of an increased risk of FSA in the subject.

2. The method according to claim 1, wherein said two or more metabolites comprise 5alpha- androstan-3alpha,17alpha-diol disulfate.

3. The method according to claim 1 or claim 2, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC.

4. The method according to any one of the preceding claims, wherein said two or more metabolites comprise 1,5-anhydroglucitol.

5. The method according to any one of the preceding claims, wherein said two or more metabolites comprise Nl,N12-diacetylspermine.

6. The method according to any one of the preceding claims, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol.

7. The method according to any one of the preceding claims, wherein said two or more metabolites comprise 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine.

8. The method according to any one of the preceding claims, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan- 3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine.

9. The method according to any one of the preceding claims, wherein said two or more metabolites comprise 4-androsten-3beta,17beta-diol monosulfate (2).

10. The method according to any one of the preceding claims, wherein said two or more metabolites comprise Acisoga.

11. The method according to any one of the preceding claims, wherein said two or more metabolites comprise Estriol 3-sulfate.

12. The method according to any one of the preceding claims, wherein said two or more metabolites comprise 4-cholesten-3-one.

13. The method according to any one of the preceding claims, wherein said two or more metabolites comprise cotinine N-oxide.

14. The method according to any one of the preceding claims, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine.

15. The method according to any one of the preceding claims, wherein said two or more metabolites comprise 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine.

16. The method according to any one of the preceding claims, wherein the level of said two or more metabolites comprises the combination of the level of two or more of l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide.

17. The method according to any one of the preceding claims, wherein the level of said two or more metabolites comprises the combination of the level of two or more of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

18. The method according to any one of the preceding claims, the method comprising applying a mathematical model to the level of said two or more metabolites.

19. The method according to claim 18, wherein the mathematical model comprises logistic regression.

20. The method according to any one of the preceding claims, the method comprising:

(a) quantifying in the biological sample the level of one or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and the level of one or more metabolites selected from the list consisting of: 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one;

(b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as:

(i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, divided by

(ii) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample; and

(c) comparing said subject metabolite ratio with a reference metabolite ratio; wherein a difference between the subject metabolite ratio and the reference metabolite ratio is indicative of an increased risk of FSA in the subject.

21. The method according to any one of claims 1-19, the method comprising:

(i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, divided by

(ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample; and

22. The method according to any one of claims 1-20, the method comprising:

(a) quantifying in the biological sample the level of metabolites l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and N1,N12- diacetylspermine;

(i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample, divided by

(ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample; and

23. The method according to any one of claims 1-19 or 21, the method comprising:

(i) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in the biological sample, divided by

(ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and

24. The method according to any one of claims 1-20, the method comprising:

(a) quantifying in the biological sample the level of metabolites l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine;

(ii) the combination of the level of Estriol 3-sulfate and the level of N1,N12- diacetylspermine in the biological sample; and

25. The method according to any one of claims 1-19 or 21, the method comprising:

(i) the combination of the level of Estriol 3-sulfate and the level of N1,N12- diacetylspermine in the biological sample, divided by (ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5-anhydroglucitol in the biological sample; and

26. The method according to any one of claims 1-20, the method comprising:

(a) quantifying in the biological sample the level of metabolites 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine;

(i) the level of 1,5-anhydroglucitol in the biological sample, divided by

27. The method according to any one of claims 1-19 or 21, the method comprising:

(i) the combination of the level of Estriol 3-sulfate and the level of N1,N12- diacetylspermine in the biological sample, divided by

(ii) the level of 1,5-anhydroglucitol in the biological sample; and

28. The method according to any one of claims 1-20 or 22, the method comprising:

(a) quantifying in the biological sample the level of metabolites l-(l-enyl-stearoyl)-2-oleoyl- GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2) and Acisoga;

(b) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5- anhydroglucitol, the level of 4-androsten-3beta,17beta-diol monosulfate (2), and the level of Acisoga in the biological sample, divided by

29. The method according to any one of claims 1-19, 21 or 23, the method comprising:

(ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC, the level of 1,5- anhydroglucitol, the level of 4-androsten-3beta,17beta-diol monosulfate (2), and the level of Acisoga in the biological sample; and

30. The method according to any one of the preceding claims, wherein said method is preceded by a step of obtaining the biological sample from the subject.

31. The method according to any preceding claim, wherein the biological sample is whole blood, blood serum, plasma, or an extract or purification therefrom, or dilution thereof.

32. The method according to any preceding claim, wherein the biological sample is obtained from a subject at least 20 weeks gestational age, optionally wherein the biological sample is obtained from a subject at least 28 weeks gestational age, optionally wherein the biological sample is obtained from a subject at least 36 weeks gestational age.

33. The method according to any preceding claim, wherein the quantifying is performed by one or more methods selected from the list consisting of: Mass spectrometry (MS), UPLC-MS/MS, SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, reverse phase (RP) liquid chromatography (LC), size permeation (gel filtration), ion exchange, affinity, FIPLC, UPLC or other LC or LC-MS-based technique, thin-layer chromatography-based analysis or a clinical chemistry analyser.

34. The method according to claim 33, wherein the quantifying is performed by MS, optionally UPLC-MS/MS.

35. The method according to claim 33 or claim 34, wherein said quantifying comprises detecting the abundance of an ion of said two or more metabolites, optionally wherein said ion is an ion of a derivative.

36. The method according to any one of claims 1-32, wherein the quantifying is performed using an immunological method, optionally Enzyme-Linked Immunosorbent Assay (ELISA).

37. The method according to claim 36, wherein said quantifying comprises detecting the level of antibody-metabolite complex.

38. The method according to any one of claims 1-32, wherein the quantifying is performed using a biosensor or a microanalytical, microengineered, microseparation or immunochromatography system.

39. The method according to any one of the preceding claims, wherein the method further comprises estimating fetal weight by fetal ultrasound.

40. The method according to any one of the preceding claims, wherein the method further comprises estimating fetal weight by assessing fundal height.

41. The method according to any one of the preceding claims, wherein the method further comprises assessing maternal or fetal or umbilical blood flow by Doppler ultrasound.

42. The method according to any one of the preceding claims, wherein upon identifying a subject as having an increased risk of FSA, monitoring of the subject is enhanced e.g. by increased frequency of fetal assessment by ultrasound.

43. A method of treating a subject identified as having an increased risk of FSA, the method comprising administering a corticosteroid medicine to the subject, wherein the subject was identified as having an increased risk of FSA by the method according to any one of the preceding claims .

44. A method of treating a subject identified as having an increased risk of FSA, the method comprising artificially inducing labour in the subject, wherein the subject was identified as having an increased risk of FSA by the method according to any one of the preceding claims.

45. The method according to claim 44, wherein artificially inducing labour comprises administering an induction agent to the subject, such as pharmacological or hormonal induction agent.

46. The method according to claim 44 or claim 45, wherein artificially inducing labour comprises rupturing the amniotic sac.

47. The method according to any one of claims 44-46, wherein artificially inducing labour comprises promoting ripening of the cervix through the use of a medical device.

48. A method of treating a subject identified as having an increased risk of FSA, the method comprising performing a caesarean section on the subject, wherein the subject was identified as having an increased risk of FSA by the method according to any one of the preceding claims.

49. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to compare:

(i) the level of two or more metabolites in a biological sample obtained from a subject, wherein said two or more metabolites comprise two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; with

(ii) reference level of said two or more metabolites.

50. A computer program according to claim 49, the computer program comprising instructions which, when the program is executed by a computer, cause the computer to apply a mathematical model to the level of said two or more metabolites.

51. The computer program according to claim 50, wherein the mathematical model comprises logistic regression.

52. The computer program according to any one of claims 49-51, wherein the level of said two or more metabolites comprises the combination of the level of two or more of l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide.

53. The computer program according to any one of claims 49-51, wherein the level of said two or more metabolites comprises the combination of the level of two or more of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

54. The computer program according to any one of claims 49-53, wherein said program further causes the computer to provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of FSA in the subject when a difference exists between the level or combination of levels of said two or more metabolites in the biological sample and the reference level or combination of reference levels of said two or more metabolites; or

(ii) indicates no increased risk of FSA in the subject when a difference does not exist between the level or combination of levels of said two or more metabolites in the biological sample and the reference level or combination of reference levels of said two or more metabolites.

55. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(i) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in a biological sample obtained from a subject, or the combination of the level of two or more of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in said biological sample, divided by

(ii) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in said biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in said biological sample.

56. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(i) the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample obtained from a subject, or the combination of the level of two or more of 5alpha-androstan- 3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3- one in said biological sample, divided by

(ii) the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in said biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in said biological sample.

57. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5- anhydroglucitol in a biological sample obtained from a subject, divided by

(ii) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in said biological sample.

58. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(i) the combination of the level of 5alpha-androstan-3alpha,17alpha-diol disulfate and the level of Nl,N12-diacetylspermine in a biological sample obtained from a subject, divided by

(ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of 1,5- anhydroglucitol in said biological sample.

59. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(ii) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in said biological sample.

60. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as: (i) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in a biological sample obtained from a subject, divided by

61. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(i) the level of 1,5-anhydroglucitol in a biological sample obtained from a subject, divided by

62. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to calculate a subject metabolite ratio as:

(i) the combination of the level of Estriol 3-sulfate and the level of Nl,N12-diacetylspermine in a biological sample obtained from a subject, divided by

(ii) the level of 1,5-anhydroglucitol in said biological sample.

63. The computer program according to any one of claims 55-62, wherein said program further causes the computer to:

(A) compare the subject metabolite ratio with a reference metabolite ratio; and

(B) provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of FSA in the subject when a difference exists between the subject metabolite ratio and a reference metabolite ratio; or

(ii) indicates no increased risk of FSA in the subject when a difference does not exist between the subject metabolite ratio and a reference metabolite ratio.

64. Use of two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4- cholesten-3-one and cotinine N-oxide, for the identification of an increased risk of FSA in a subject.

65. The use according to claim 64, wherein said two or more metabolites comprise 5alpha- androstan-3alpha,17alpha-diol disulfate .

66. The use according to claim 64 or claim 65, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC.

67. The use according to any one of claims 64-66, wherein said two or more metabolites comprise 1,5-anhydroglucitol.

68. The use according to any one of claims 64-67, wherein said two or more metabolites comprise Nl,N12-diacetylspermine.

69. The use according to any one of claims 64-68, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC and 1,5-anhydroglucitol.

70. The use according to any one of claims 64-69, wherein said two or more metabolites comprise 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine.

71. The use according to any one of claims 64-70, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha- diol disulfate and Nl,N12-diacetylspermine.

72. The use according to any one of claims 64-71, wherein said two or more metabolites comprise 4-androsten-3beta,17beta-diol monosulfate (2).

73. The use according to any one of claims 64-72, wherein said two or more metabolites comprise Acisoga.

74. The use according to any one of claims 64-73, wherein said two or more metabolites comprise Estriol 3-sulfate .

75. The use according to any one of claims 64-74, wherein said two or more metabolites comprise 4-cholesten-3-one.

76. The use according to any one of claims 64-75, wherein said two or more metabolites comprise cotinine N-oxide.

77. The use according to any one of claims 64, 66-69 or 74, wherein said two or more metabolites comprise l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine.

78. The use according to any one of claims 64, 67, 68 or 74, wherein said two or more metabolites comprise 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine.

79. A kit comprising (i) reagents and/or a biosensor capable of detecting and/or quantifying two or more metabolites selected from the list consisting of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide; and (ii) instructions for use in screening for an increased risk of FSA.

80. An ion of two or more of the following metabolites: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5- anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide, for use in screening for an increased risk of FSA, optionally wherein the ion is an ion of a derivative.

81. A method of screening for an increased risk of FSA in a subject or a predisposition thereto, said method comprising: (a) obtaining a biological sample from the subject;

(b) detecting and/or quantifying in the biological sample two or more metabolites selected from the list consisting of: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga, Estriol 3-sulfate, 4-cholesten-3-one and cotinine N-oxide by:

(i) ionising the biological sample or a fraction thereof, optionally wherein the sample is derivatised prior to ionising; and

(ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of said two or more metabolites; and

(c) identifying an increased risk of FSA in the subject when the level of said two or more metabolites in the biological sample is different from the reference level of said two or more metabolites.

82. The method according to claim 81, the method comprising:

(a) obtaining a biological sample from the subject;

(b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by:

(ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of one or more of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga, and cotinine N-oxide; and one or more of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one;

(c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as:

(d) identifying an increased risk of FSA in the subject when the subject metabolite ratio is different from the reference metabolite ratio.

83. The method according to claim 81, the method comprising:

(a) obtaining a biological sample from the subject;

84. The method according to claim 81 or claim 82, the method comprising:

(a) obtaining a biological sample from the subject;

(b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine by:

(ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine;

(c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of

1,5-anhydroglucitol in the biological sample, divided by

85. The method according to claim 81 or claim 83, the method comprising:

(a) obtaining a biological sample from the subject;

(ii) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of

1,5-anhydroglucitol in the biological sample; and

86. The method according to claim 81 or claim 82, the method comprising:

(a) obtaining a biological sample from the subject;

(b) detecting and/or quantifying in the biological sample metabolites l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine by:

(ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine;

(i) the combination of the level of l-(l-enyl-stearoyl)-2-oleoyl-GPC and the level of

1,5-anhydroglucitol in the biological sample, divided by (ii) the combination of the level of Estriol 3-sulfate and the level of N1,N12- diacetylspermine in the biological sample; and

87. The method according to claim 81 or claim 83, the method comprising:

(a) obtaining a biological sample from the subject;

88. The method according to claim 81 or claim 82, the method comprising:

(a) obtaining a biological sample from the subject;

(b) detecting and/or quantifying in the biological sample metabolites 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine by:

(ii) detecting and/or quantifying ion(s) or ion(s) of derivatives of: 1,5- anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine;

(i) the level of 1,5-anhydroglucitol in the biological sample, divided by

89. The method according to claim 81 or claim 83, the method comprising:

(a) obtaining a biological sample from the subject;

(ii) the level of 1,5-anhydroglucitol in the biological sample; and

90. A method of screening for risk of FSA in a subject, said method comprising:

(a) obtaining a biological sample from a subject;

(i) contacting the biological sample with antibodies against said two or more metabolites; and

(ii) detecting and/or quantifying binding between said two or more metabolites and their respective antibodies; and

(c) identifying an increased risk of FSA in the subject when the level of said two or more metabolites in the biological sample from the subject is different from the reference level of said two or more metabolites.

91. The method according to claim 90, the method comprising:

(a) obtaining a biological sample from a subject;

(b) detecting and/or quantifying in the biological sample one or more of l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one by:

(i) contacting the biological sample with an antibodies against one or more of 1-(1- enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one; and

(ii) detecting and/or quantifying binding between one or more of l-(l-enyl-stearoyl)- 2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; and one or more of 5alpha-androstan-3alpha,17alpha- diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one and their respective antibodies;

92. The method according to claim 90, the method comprising:

(a) obtaining a biological sample from a subject;

93. The method according to claim 90 or claim 91, the method comprising:

(a) obtaining a biological sample from a subject;

(i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and

(ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and N1,N12- diacetylspermine and their respective antibodies;

94. The method according to claim 90 or claim 92, the method comprising:

(a) obtaining a biological sample from a subject;

(i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and Nl,N12-diacetylspermine; and (ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC,

1,5-anhydroglucitol, 5alpha-androstan-3alpha,17alpha-diol disulfate and N1,N12- diacetylspermine and their respective antibodies;

1,5-anhydroglucitol in the biological sample; and

95. The method according to claim 90 or claim 91, the method comprising:

(a) obtaining a biological sample from a subject;

(i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine; and

(ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC,

1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine and their respective antibodies;

1,5-anhydroglucitol in the biological sample, divided by

96. The method according to claim 90 or claim 92, the method comprising:

(a) obtaining a biological sample from a subject;

(i) contacting the biological sample with antibodies against: l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine; and (ii) detecting and/or quantifying binding between: l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine and their respective antibodies;

97. The method according to claim 90 or claim 91, the method comprising:

(a) obtaining a biological sample from a subject;

(i) contacting the biological sample with antibodies against: 1,5-anhydroglucitol, Estriol 3-sulfate and Nl,N12-diacetylspermine; and

(ii) detecting and/or quantifying binding between: 1,5-anhydroglucitol, Estriol 3- sulfate and Nl,N12-diacetylspermine and their respective antibodies;

(i) the level of 1,5-anhydroglucitol in the biological sample, divided by

98. The method according to claim 90 or claim 92, the method comprising:

(a) obtaining a biological sample from a subject;

(c) determining the subject metabolite ratio, wherein the subject metabolite ratio is defined as: (i) the combination of the level of Estriol 3-sulfate and the level of N1,N12- diacetylspermine in the biological sample, divided by

(ii) the level of 1,5-anhydroglucitol in the biological sample; and

99. The method or computer program according to any one of the preceding claims, wherein the combination of the level of said two or more metabolites comprises:

(a) the sum of the level of said two or more metabolites;

(b) the product of the level of said two or more metabolites; and/or

(c) the result of any other mathematical operation applied to the level of said two or more metabolites, wherein application of said mathematical operation yields a value greater than the individual values entered into the operation; and/or

(d) any combination thereof.

100. The method according to any one of claims 1-48 or 81-99, the computer program according to any one of claims 49-63 or 99, the use according to any one of claims 64-78, the kit according to claim 79 or the ion according to claim 80, wherein the FSA is fetal growth restriction (FGR).

101. The method according to any one of claims 1-48 or 81-99, the computer program according to any one of claims 49-63 or 99, the use according to any one of claims 64-78, the kit according to claim 79 or the ion according to claim 80, wherein the FSA is large for gestational age (LGA).

102. The method according to any one of claims 1, 3, 4, 6, 8-10 or 13-16, wherein a greater level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-

3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample compared to the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-

3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide, or the combination of the reference levels of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide is indicative of an increased risk of FGR in the subject.

103. The method according to any one of claims 1, 3, 4, 6, 8-10 or 13-16, wherein a lower level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-

3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide, or the combination of the reference levels of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide is indicative of an increased risk of LGA in the subject.

104. The method according to any one of claims 1, 2, 5, 7, 8, 11, 12, 14, 15 or 17, wherein a lower level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample compared to the reference level of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten- 3-one, or the combination of the reference levels of two or more of 5alpha-androstan-

3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one is indicative of an increased risk of FGR in the subject.

105. The method according to any one of claims 1, 2, 5, 7, 8, 11, 12, 14, 15 or 17, wherein a greater level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3- sulfate and 4-cholesten-3-one in the biological sample compared to the reference level of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten- 3-one, or the combination of the reference levels of two or more of 5alpha-androstan-

3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one is indicative of an increased risk of LGA in the subject.

106. The method according to any one of claims 20, 22, 24, 26, 28, 82, 84, 86, 88, 91, 93, 95 or 97, wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

107. The method according to any one of claims 20, 22, 24, 26, 28, 82, 84, 86, 88, 91, 93, 95 or 97, wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

108. The method according to any one of claims 21, 23, 25, 27, 29, 83, 85, 87, 89, 92, 94, 96 or 98, wherein a lower subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of FGR in the subject.

109. The method according to any one of claims 21, 23, 25, 27, 29, 83, 85, 87, 89, 92, 94, 96 or 98, wherein a greater subject metabolite ratio than the reference metabolite ratio is indicative of an increased risk of LGA in the subject.

110. The computer program according to any one of claims 49-52, wherein said program further causes the computer to provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of FGR in the subject when the level of one of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta- diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample is greater than the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide, or the combination of the reference levels of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; or (ii) indicates no increased risk of FSA in the subject when a difference does not exist between the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC,

1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide in the biological sample and the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC,

1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N- oxide, or the combination of the reference levels of two or more of l-(l-enyl-stearoyl)-2- oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide.

111. The computer program according to any one of claims 49-52, wherein said program further causes the computer to provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of LGA in the subject when the level of one of l-(l-enyl- stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten-3beta,17beta- diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample is lower than the reference level of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4-androsten- 3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide, or the combination of the reference levels of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide; or

(ii) indicates no increased risk of FSA in the subject when a difference does not exist between the level of one of l-(l-enyl-stearoyl)-2-oleoyl-GPC, 1,5-anhydroglucitol, 4- androsten-3beta,17beta-diol monosulfate (2), Acisoga and cotinine N-oxide in the biological sample, or the combination of the level of two or more of l-(l-enyl-stearoyl)-2-oleoyl-GPC,

112. The computer program according to any one of claims 49-51 or 53, wherein said program further causes the computer to provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of FGR in the subject when the level of one of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample is lower than the reference level of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one, or the combination of the reference levels of two or more of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; or (ii) indicates no increased risk of FSA in the subject when a difference does not exist between the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample and the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one, or the combination of the reference levels of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

113. The computer program according to any one of claims 49-51 or 53, wherein said program further causes the computer to provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of LGA in the subject when the level of one of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample is greater than the reference level of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one, or the combination of the reference levels of two or more of 5alpha- androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4- cholesten-3-one; or

(ii) indicates no increased risk of FSA in the subject when a difference does not exist between the level of one of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample, or the combination of the level of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, Nl,N12-diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one in the biological sample and the reference level of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one, or the combination of the reference levels of two or more of 5alpha-androstan-3alpha,17alpha-diol disulfate, N1,N12- diacetylspermine, Estriol 3-sulfate and 4-cholesten-3-one.

114. The computer program according to any one of claims 55, 57, 59 or 61, wherein said program further causes the computer to:

(A) compare the subject metabolite ratio with a reference metabolite ratio; and

(B) provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of FGR in the subject when the subject metabolite ratio is greater than the reference metabolite ratio; or

115. The computer program according to any one of claims 55, 57, 59 or 61, wherein said program further causes the computer to:

(A) compare the subject metabolite ratio with a reference metabolite ratio; and (B) provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of LGA in the subject when the subject metabolite ratio is lower than the reference metabolite ratio; or

116. The computer program according to any one of claims 56, 58, 60 or 62, wherein said program further causes the computer to:

(A) compare the subject metabolite ratio with a reference metabolite ratio; and

(B) provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of FGR in the subject when the subject metabolite ratio is lower than the reference metabolite ratio; or

117. The computer program according to any one of claims 56, 58, 60 or 62, wherein said program further causes the computer to:

(A) compare the subject metabolite ratio with a reference metabolite ratio; and

(B) provide a diagnostic report, wherein the diagnostic report:

(i) indicates an increased risk of LGA in the subject when the subject metabolite ratio is greater than the reference metabolite ratio; or

118. The method according to any one of claims 1-48 or 81-109, wherein no difference between the level of metabolites or the combination of levels of metabolites in the biological sample and the reference level of said metabolites or the combination of reference levels of said metabolites, or between the subject metabolite ratio and the reference metabolite ratio is indicative of no increased risk of FSA.

119. The method according to any one of claims 1-48 or 81-109, the computer program according to any one of claims 49-63 or 110-117, the use according to any one of claims 64-78, the kit according to claim 79 or the ion according to claim 80, wherein risk of FSA is the risk of FSA occurring at term.

120. The method according to any one of claims 100, 102, 104, 106 or 108, or the computer program according to any one of claims 110, 112, 114 or 116, wherein risk of FGR is the risk of FGR occurring at term.

121. The method according to any one of claims 101, 103, 105, 107 or 109, or the computer program according to any one of claims 111, 113, 115 or 117, wherein risk of LGA is the risk of LGA occurring at term.