Field of the Art

[001] The present disclosure relates generally to methods of detecting or determining aging and/or oxidative damage in tissue, such as placental tissue, skin, kidney and brain tissue. The disclosure also relates to methods of delaying, slowing or preventing aging and/or oxidative damage in such tissue.

Background

[002] Accumulated random damage to tissue may affect tissue functionality and contribute to aging. Metabolic processes and growth of cells generate reactive oxygen species that may cause damage to proteins, lipids and nucleic acids. While cells possess defence mechanisms against oxidative stress, such as enzymes like catalase and superoxide dismutase, if the repair mechanisms are inadequate to compensate for the generation of reactive oxygen species, damage and aging of the tissue may occur.

[003] Growth pathways are generally associated with increased oxidative stress. Due to the placenta's function in promoting the growth of a fetus it is not surprising that indicators of oxidative damage can be found in the placenta, in particular nearing the end of pregnancy when fetal growth is rapid. The inventors have hypothesised that stillbirth may be related to a reduced ability of the ageing placenta to sustain the fetus (Smith et al 2013, Placenta, 34:310-313). Supporting this view a histopathological study of placentas associated with cases of unexplained intrauterine death at term revealed that 91% showed thickening of the maternal spiral artery walls, 54% contained placental infarcts, 10% had calcified areas and 13% demonstrated vascular occlusion; changes that are associated with aging in other organs. The inventors demonstrated that placentas from women who delivered late or had stillbirth had biochemical evidence of aging and oxidative stress.

[004] Unexplained fetal death is a common complication of pregnancy occurring in approximately 1 in 200 pregnancies in developed countries and more frequently in the developing world. Unexplained fetal death is 40 times more common than sudden infant death syndrome but attracts far less attention, yet for the families involved the event is devastating. While no cause has been established, stillbirth may be associated with fetal growth restriction, congenital abnormality, antepartum haemorrhage, perinatal infection, pre-eclampsia and maternal factors such as maternal height and age, obesity, hypertension, diabetes, smoking, parental consanguinity, ethnicity and country of birth. While there are risk factors for stillbirth, the cause of many cases remains unexplained.

[005] In some antepartum cases, lack of movement of the fetus may occur prior to stillbirth, although there may be no warning signs. The lack of understanding of the cause of stillbirth makes prediction of risk during pregnancy difficult. Stillbirth can be defined as death of a fetus before or during birth at more than or equal to 20 weeks gestation or of more than or equal to 400 g birthweight (Perinatal Society of Australia and New Zealand Clinical Practice Guideline for Perinatal Mortality; Second Edition, version 2.2, April 2009). Identifying indicators of risk of stillbirth may provide an opportunity for pregnant women to undergo prophylactic caesarean section or induction of labour to reduce risk to the fetus. Therefore prognostic biomarkers for stillbirth risk would be highly beneficial in prevention of stillbirth.

[006] The optimal length of a pregnancy is likely to differ between individuals. While a baby may be viable if born preterm, complications are likely if adequate development has not occurred in utero. In general a longer gestation (up to approximately 41 weeks) is associated with better development of a baby's anatomy and physiological systems. Indeed, infants born at 41 weeks gestation are less likely to require special education later in life than infants born at 37 weeks gestation. However, a prolonged gestational period can result in post-maturity symptoms, where the fetus continues to grow and to require additional supplies of nutrients. Post-maturity syndrome is a condition seen in post-dated infants who show evidence of late gestation failure of nutrition. Normal human infants born at term have 12-14% body fat whereas post maturity syndrome is associated with the birth of a baby with severe wrinkling of the skin due to loss of subcutaneous fat. Post maturity syndrome is rarely seen in modern obstetric practice where delivery is usually affected before 42 weeks of gestation using induction of labour or caesarean section if labour has not occurred spontaneously. Notably, the risk of stillbirth is significantly increased in post-term pregnancies, the rate rising exponentially as gestation progresses beyond 38 weeks. Therefore there appears to be a window of optimal gestation in pregnancy, however this window is likely to differ between individuals. There is currently no way to determine the ideal gestational window for individual pregnancies. While placental ageing may be an indicator of an appropriate time to deliver a baby, there is currently no method to determine the oxidative state of the placenta in a pregnant woman with minimal risk to the fetus.

Summary of the Disclosure

[007] The present disclosure is predicated on the inventors' surprising discovery that aldehyde oxidase 1 (AOXl) expression and activity (and levels of markers thereof such as 4- hydroxynonenal, 4HNE) correlates with tissue aging and oxidative damage in multiple tissue including placenta and kidney. The inventors have also demonstrated that oxidative stress in the placenta may be reduced by the administration of inhibitors of AOXl such as raloxifene, or G- protein-coupled estrogen receptor 1 (GPER1) agonists, such as Gl. As exemplified herein inhibition of AOXl prevents oxidative and lysosomal changes in placental tissue.

[008] The inventors therefore have identified an effective biomarker that may be non-invasively sampled to give an indication of placental oxidative stress, thereby indicating that a pregnancy may be at risk of stillbirth. Oxidative and lysosomal changes similar to those observed in the placenta can also be found in diseases associated with ageing (e.g. Alzheimer's disease, Huntington's disease). Accordingly, AOXl also provides a marker of, and a target for treatment or prevention of, oxidative damage and ageing in multiple tissue types.

[009] A first aspect of the present disclosure provides a method for detecting or determining aging in body tissue, the method comprising measuring one or more markers of aldehyde oxidase 1 (AOXl) expression or activity in a biological sample obtained from a subject, wherein the level of AOXl expression or activity, as determined by the level(s) of the one or more markers, is indicative of aging in the tissue.

[010] In an embodiment, the tissue is selected from placental tissue, skin, kidney or brain tissue.

[011] In an exemplary embodiment, the subject is pregnant and the tissue is placental tissue.

[012] The biological sample may be, for example, a sample of the relevant tissue, or a fluid sample, such as blood (whole blood, plasma or serum), urine, saliva or amniotic fluid. In embodiments in which the subject is pregnant, the sample may comprise, for example, maternal blood, fetal blood, urine, saliva or amniotic fluid. In embodiments in which the subject is pregnant, the biological sample is typically of maternal origin, optionally maternal blood. In other embodiments in which the subject is pregnant, the biological sample may comprise a derivative of a sample from the pregnant subject, placental tissue and/or a fetus, such as total RNA derived from maternal blood, where the total RNA may be maternal, placental or fetal in origin.

[013] The one or more markers of AOX 1 expres sion or activity may be measured from material released from aging tissue into the blood. In an embodiment in which the subject is pregnant, the one or more markers of AOXl expression or activity may be measured from material released from aging placental tissue into the maternal blood. The one or more markers may therefore be measured in maternal blood. In exemplary embodiments, the one or more markers are derived from free RNA or DNA, fragments of placental cells, exosomes or microparticles present within the biological sample.

[014] Expression of AOXl may be measured at the mRNA or gene level, or at the polypeptide or protein level. In exemplary embodiments, a suitable marker of AOXl expression or activity may comprise AOXl mRNA. In other embodiments, a suitable marker of AOXl expression or activity may comprise a molecule downstream of AOXl, such as a lipid peroxidation product. Exemplary lipid peroxidation products are 4-hydroxynonenal (4HNE) and malondi aldehyde (MDA). Other products of oxidative damage that may be measured include 8-hydroxy- deoxyguanosine (80HdG), 8-hydroxyguanosine (8HOG). In exemplary embodiments, any two or more markers of AOX 1 expression or activity may be determined or measured in combination. For example, AOXl mRNA may be measured in combination with a lipid peroxidation product such as 4HNE.

[015] Typically, the level of expression or activity of AOXl in a subject, as determined by the one or more markers, is compared to a reference value or values of AOX 1 expression or activity, or one or more markers thereof, in tissue in which oxidative damage is not present. A reference value may be obtained or derived from one or more control or reference individuals. Alternatively, a reference value may be derived from one or more biological samples obtained from the subject at different time points which may be used as a baseline value to which a further biological sample from the subject may be compared. A person skilled in the art would be familiar with ways of determining and selecting an appropriate reference sample. [016] Typically an increased level of expression or activity of AOXl, or one or more markers thereof, in a biological sample from a subject compared to a reference value is indicative of oxidative stress and/or aging in the tissue. In exemplary embodiments, the tissue is placental tissue and the biological sample is maternal blood.

[017] In particular embodiments, additional markers of oxidative stress, such as placental or fetal oxidative stress, may also be measured in the biological sample, or in a further biological sample. Non-limiting examples of such additional oxidative damage or stress markers may include AOXl protein, glutathione, or protein carbonyls.

[018] In embodiments in which the subject is pregnant, the method may be carried out one or more times at any time during pregnancy, in particular in the latter half of the pregnancy, such as from approximately 20 weeks gestation in the case of a human subject. In particular embodiments the method may be carried out in a post-dated pregnancy, for example after 40 weeks gestation in the case of a human subject.

[019] A specific application of a method of the present disclosure is to detect aging in the placenta of a pregnant subject in order to predict the risk of stillbirth, wherein an increased level of expression or activity of AOXl, or one or more markers thereof, in a biological sample compared to a reference value(s) is indicative of placental aging and the risk of stillbirth. Aging of the placenta may be associated with placental and/or fetal oxidative damage, reduced fetal health and increased risk of stillbirth. Placental oxidative stress and placental aging may also be associated with inadequate nutrient transfer to a fetus. The method may also be used to diagnose pre-eclampsia in a pregnant subject or to diagnose intra-uterine growth restriction in a fetus. The method may also be used to predict the risk of, or associated with, a post-dated pregnancy.

[020] A further embodiment of the present disclosure comprises obtaining a biological sample from a pregnant subject and measuring one or more markers of placental AOXl expression or activity in the biological sample in order to determine the need for intervention to prevent stillbirth in a pregnancy, wherein increased AOXl expression or activity, or levels of one or more markers thereof, compared to a reference value(s) indicates intervention may be required.

Such interventions may include prophylactic caesarean section, induction of labour and/or administration of a therapy to prevent or delay placenta ageing or prolong viability of the fetus in utero. Therefore, in an embodiment, the method may be used to determine an appropriate time to deliver a baby, by determining whether placental oxidative stress is at a level where the fetus is at risk of stillbirth. Increased AOXl expression or activity, or levels of one or more markers thereof, compared to a reference value(s) may be suggestive or indicative of an appropriate time to deliver a baby.

[021 ] In an embodiment, a method of the disclosure may be used to monitor the risk of stillbirth during a pregnancy by detecting or determining aging in placental tissue by measuring one or more markers of AOXl expression or activity in one or more biological samples obtained from a subject over time. In such a method, biological samples are obtained from a pregnant subject at multiple time points during the pregnancy, for example on a fortnightly, weekly or daily basis, and one or more markers of placental AOXl expression or activity are measured in each biological sample from each time point. An increase in AOXl expression or activity, or levels of one or more markers thereof, over time may be indicative of increased risk of stillbirth, and thus may be indicative of the need for intervention to prevent stillbirth.

[022] A second aspect of the present disclosure provides a method for detecting placental aging, the method comprising obtaining a biological sample from a pregnant subject and measuring one or more markers of placental AOXl expression or activity in the biological sample, wherein the level of AOXl expression or activity or of the one or more markers thereof is indicative of placental aging.

[023] Typically increased AOXl expression or activity, or levels of one or more markers thereof, compared to a reference value(s) indicates aging in the placenta. A suitable reference value may be derived or obtained from a sample from tissue of one or more individuals in which placental tissue does not exhibit signs of oxidative damage, or may be derived or obtained from the subject at an earlier time point during the pregnancy.

[024] The placental aging may be associated with increased risk of stillbirth, with preeclampsia, with intra-uterine growth restriction in the fetus, and/or with an increased risk of a post-dated pregnancy.

[025] A third aspect of the present disclosure provides a method for predicting the risk of stillbirth in a pregnancy, the method comprising obtaining a biological sample from a pregnant subject and measuring one or more markers of placental AOXl expression or activity in the biological sample, wherein the level of AOX1 expression or activity, or of the one or more markers thereof, is indicative of risk of stillbirth.

[026] The risk of stillbirth may be determined directly or indirectly. For example the level of AOX1 expression or activity, or of the one or more markers thereof, may be indicative of preeclampsia, with intra-uterine growth restriction in the fetus, and/or with an increased risk of postdated pregnancy, each of these being indicators or factors associated with an increased risk of stillbirth.

[027] Typically increased AOX1 expression or activity, or levels of one or more markers thereof, compared to a reference value(s) is indicative of stillbirth risk. A reference value may be obtained or derived from one or more control or reference individuals. A suitable reference value may be derived or obtained from a sample from tissue of one or more individuals in which placental tissue do not exhibit signs of oxidative damage, or may be derived or obtained from the subject at an earlier time point during the pregnancy.

[028] Accordingly, a fourth aspect of the present disclosure provides a method for determining the need for intervention to prevent post-dated pregnancy or stillbirth in a pregnancy, the method comprising obtaining a biological sample from a pregnant subject and measuring one or more markers of placental aldehyde oxidase 1 (AOX1) expression or activity in the biological sample, wherein the level of AOX1 expression or activity, or of the one or more markers thereof, is indicative of the need for intervention.

[029] Intervention may comprise at least one of prophylactic caesarean section, induction of labour or administration of a therapy to delay placenta aging or prolong viability of the fetus in utero. Where the fetus is too premature to deliver safely or is too underdeveloped to be viable, an intervention may comprise treatment of the subject to reduce oxidative stress or AOX1 expression or activity as discussed in further aspects and embodiments of the present disclosure.

[030] A fifth aspect of the present disclosure provides a method for detecting placental oxidative stress, the method comprising obtaining a biological sample from a pregnant subject and measuring one or more markers of placental AOX1 expression or activity in the biological sample, wherein the level of AOX1 expression or activity, or of the one or more markers thereof, is indicative of placental oxidative stress. [031] Typically increased AOX1 expression or activity, or levels of one or more markers thereof, compared to a reference value(s) is indicative of placental oxidative stress. A reference value may be obtained or derived from one or more control or reference individuals. A suitable reference value may be derived or obtained from a sample from tissue of one or more individuals in which placental tissue do not exhibit signs of oxidative damage, or may be derived or obtained from the subject at an earlier time point during the pregnancy.

[032] A sixth aspect of the present disclosure provides a method for monitoring placental aging, placental oxidative stress or the risk of post-dated pregnancy or stillbirth over the course of a pregnancy, the method comprising: (i) obtaining a biological sample from a pregnant subject at a first time point during the course of a pregnancy; (ii) measuring one or more markers of placental AOX1 expression or activity in the biological sample; (iii) obtaining one or more further biological samples from the subject at one or more later time points during the pregnancy; and (iv) measuring one or more markers of placental AOX1 expression or activity in the one or more further biological samples, wherein an increase in the level of AOX 1 expression or activity, or of the one or more markers thereof, over time is indicative of placental aging, placental oxidative stress or the risk of post-dated pregnancy or stillbirth.

[033] Measurement of the one or more markers may begin, for example, in mid gestation. In an exemplary embodiment biological samples may be obtained from about 20 weeks gestation. Intervals between the time points may be determined by a person skilled in the art and may be daily, weekly, fortnightly, monthly, or another appropriate time period determined by a practitioner. The time points may increase in frequency throughout the course of pregnancy, particularly at later gestational ages.

[034] In a seventh aspect of the present disclosure there is provided a method of slowing or preventing aging or oxidative damage in one or more cells or tissues, the method comprising exposing the one or more cells or tissues to an inhibitor of AOX1.

[035] The inhibitor may act directly or indirectly on AOX1. In an exemplary embodiment, the inhibitor of AOX1 is raloxifene or a G protein-coupled estrogen receptor 1 (GPER1) agonist. An exemplary GPER1 agonist is G-l ((l-[4-(6-bromobenzo[ l,3] dioxol-5yl)-3a,4,5,9b- tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone).

[036] In an exemplary embodiment the tissue is placental tissue. Accordingly, the inhibitor of AOX1 is administered to a pregnant subject.

[037] In a further exemplary embodiment, the tissue is skin, kidney or brain tissue.

[038] In an eighth aspect of the present disclosure there is provided a method of treating a disease or condition associated with aging or oxidative damage in one or more cells or tissues, the method comprising administering to a subject in need thereof an inhibitor of AOX1.

[039] In a ninth aspect of the present disclosure there is provided a method for preventing or reducing the risk of post-dated pregnancy or stillbirth, the method comprising administering to a pregnant subject an inhibitor of AOX1.

[040] Further aspects provide the use of an inhibitor or AOX1 in the manufacture of a medicament for lowing or preventing aging or oxidative damage, for treating a disease or condition associated with aging or oxidative damage, or for preventing or reducing the risk of post-dated pregnancy or stillbirth.

Brief Description of the Drawings

[041] Aspects and embodiments of the present disclosure are described herein, by way of non- limiting example only, with reference to the following drawings.

[042] Figure 1: Aldehyde oxidase regulates oxidative damage in placentas associated with stillbirth. Oxidative damage to fatty acids can be demonstrated by 4-hydroxynoneal (4HNE), which is associated with altered lysosome and/or autophagosome function. Oxidative damage can also alter nucleic acids (DNA and/or RNA) in the cell, which may be demonstrated by the presence of 8-hydroxy-guanosine (80HdG) or 8-hydroxyguanosine (8HOG). 4HNE, 80HdG and 8HOG can be indicative of ageing in the placenta. Aldehyde oxidase may be inhibited by raloxifene and by using the agonist G 1 to interact with the G protein-coupled estrogen receptor 1. [043] Figure 2: Relationship between stillbirth and number of continuing pregnancies. Kaplan Myer plot of number of continuing pregnancies as a function of gestational age and plot of unexplained stillbirths per 1000 continuing pregnancies; data from Omigbodun and Adewuyi (1997) {J Natl Med Assoc, 89: 617) and Sutanet al. (2010) (J ^' Perinatal, 30: 311-318). Plot shows the increase in risk of stillbirth with time consistent with the operational definition of aging proposed by Johnson et al (1999) {Cell, 96: 291-302) and the relatively small number of pregnancies at risk of stillbirth by 41 weeks because of prior delivery.

[044] Figure 3: DNA/RNA oxidation in post-dated and stillbirth placentas. The graph illustrates that post-dated and stillbirth placentas have increased intensity of nuclear 80HdG/8HOG staining (p<0.0001 for post-dated placentas, p=0.0005 for stillbirth placentas, Mann Whitney test) compared to term placentas. Open circles and filled circles represent term caesarean non-labouring placentas (n=10) and term vaginal delivery labouring placentas (n=8), respectively. Open squares and filled squares represent post-dated labouring caesarean placentas (n=5) and post-dated labouring vaginal delivery placentas (n=13), respectively. Filled triangles represent third trimester labouring vaginal delivery unexplained stillbirth placentas (n=4). Each point in the graph represents the average intensity of 80HdG/8HOG of 60 nuclei in 6 images per placenta photographed at 100X magnification and 1.4 optical resolution.

[045] Figure 4: Lipid peroxidation is increased in post-dated and stillbirth placentas. The intensity of 4HNE is significantly increased in post-dated placentas (p<0.0001, Mann Whitney test) and stillbirth placentas (p=0.0013, Mann Whitney test). Open circles and filled circles represent term caesarean non-labouring placentas (n=21) and term vaginal delivery labouring placentas (n=14), respectively. Open squares and filled squares represent post-dated labouring caesarean placentas (n=10) and post-dated labouring vaginal delivery placentas (n=18), respectively. Filled triangles represent third trimester labouring vaginal delivery unexplained stillbirth placentas (n=4). Each point in the graph represents the mean intensity per unit area for 6 images taken for each individual placenta photographed at 100X magnification and 1.4 optical resolution.

[046] Figure 5: Levels of 4HNE in maternal plasma from early term (n=17), late term (n=26), pre-eclamptic (PE, n=22) and intrauterine growth restriction (IUGR, n=24) cases. The late term,

PE and IUGR groups have significantly higher levels of 4HNE than early term. Early term v late term, p = 0.0016; early term v PE, p = 0.0017; early term v IUGR, p = 0.0016. [047] Figure 6: Changes in lysosomal distribution in post-dated and stillbirth placentas. Immunohistochemistry of LAMP2, a lysosomal marker showed that lysosomes predominantly localise to the apical surface of term placentas (A), whereas lysosome distribution extends to the perinuclear and basal surface of syncytiotrophoblast in post-dated (B) and stillbirth placentas (C) (e.g. as designated by white lines). Intensity calculation across the syncytiotrophoblast showed that the distribution of LAMP2 in post-dated (n=5, Figure 5F) and unexplained stillbirth placentas (n=4, Figure 5G) shifts to the perinuclear and basal surface whereas lysosome distribution in term caesarean placentas (n=5, Figure 5D) and term vaginal delivery placentas (n=5, Figure 5E) remained in the apical region of the syncytiotrophoblast. DAPI staining identifies the nuclei. In Figures 5D to 5G each coloured line represents results on an individual placenta, and shows the mean intensity of LAMP2 across the syncytiotrophoblast at 5 random sites per image (example represented by light green line in 5A, 5B and 5C) for 6 separate images per placenta. Images were photographed at 100X magnification and 1.4 optical resolution. Scale bar, 20 μ m .

[048] Figure 7: Larger autophagosomes occur in post-dated and stillbirth placentas. Autophagosome size was quantified using NIS element software and the diameter was measured at an arbitrary intensity range of 1000-3000, diameter range 0.2-1 μιη and circularity range 0.5-1. Analysis showed that post-dated and stillbirth placentas have significantly larger (p=0.012 and p=0.0019, respectively, Mann Whitney test) autophagosomes than term placentas. Open circles and filled circles represent term caesarean non-labouring placentas (n=ll) and term vaginal delivery labouring placentas (n=10), respectively. Open squares and filled squares represent post-dated labouring caesarean placentas (n=8) and post-dated labouring vaginal delivery placentas (n=15), respectively. Filled triangles represents unexplained stillbirth placentas (n=4). Each point in the graph represents the average diameter of LC3B particles in six images taken for each placenta.

[049] Figure 8: Oxidised lipids within autophagosomes of post-dated placentas. Representative dual labelled fluorescence immunostaining showed that LC3B, an autophagosome marker is co-localised with 4HNE, a marker of lipid peroxidation. Dots (pointed by arrow heads in C) indicate the co- localization. DAPI staining indicates the nuclei. Original magnifications 100X; scale bar 20 μιη. [050] Figure 9: RN A- sequencing data showed post-dated placentas have significantly higher expression of AOX1 mRNA than term placentas.

[051] Figure 10: Role of aldehyde oxidase 1 (AOX1) in the production of 4HNE. Representative dual labelled fluorescence immunostaining in post-dated (A-C) and stillbirth (D- F) placentas showed that AOX1 positive particles are co-localized with 4HNE. Dots (pointed by arrow heads in C and F) indicate the degree of co-localization. Nuclei are stained with DAPI. Real-time PCR showed that expression of AOX1 mRNA is increased in post-dated (p=0.0097) and stillbirth (p=0.012) placentas compared to term placentas (G). Original magnification 100X; scale bar 20 μ m .

[052] Figure 11: Pharmacologic inhibition of 4HNE production and expression of GPERl in placenta. The production of 4HNE (induced by serum starvation) is significantly reduced after treating placental explants with raloxifene, an AOX1 inhibitor (A), and Gl, a membrane estrogen receptor GPERl agonist (B). Data are mean + SEM, */?<0.05 (N=6).

[053] Figure 12: Changes in autophagosome size in placental explants cultured in serum deprived medium. Immunohistochemical analysis showed that the size of autophagosomes (LC3B positive particles) increased 24 h after serum starvation compared to 0 h. Data presented as mean + SEM, ***p=0.0002 (N=13).

[054] Figure 13: Detection of GPERl in different tissues by real-time PCR and western- blotting. The real-time qPCR data showed that the mRNA for GPERl is expressed in higher amounts in term placenta than in amnion, chorion and decidua (A). The amount of expression of mRNA of GPERl has the following order in these tissues: decidua<chorion<amnion<placenta

(A) . The proteins were extracted from the breast cancer cell line MCF-7, term placenta, term myometrium, term amnion, term chorion and term decidua and western-blotting was performed

(B) . Placenta, myometrium and MCF-7 cell lines expressed higher amounts of GPERl than amnion, chorion or decidua (B). Western- blotting data showed that all the tissues expressed glycosylated GPERl (denoted by ** or by ***) and non-glycosylated nascent GPERl (denoted by *). The sypro-ruby stain of same PVDF membrane is used as internal loading control (C).

[055] Figure 14: AOX1 mRNA is increased in total maternal blood in the case of stillbirth as compared to term pregnancies. [056] Figure 15: Immuno staining for 4HNE in kidney tissue from a 66 year old subject (A) and from a 21 year old subject (B). All images are at 20X magnification, unless indicated.

Detailed description

[057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, typical methods and materials are described.

[058] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[059] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[060] As used herein, the term "subject" includes any mammal, such as humans, non-human primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys, goats), laboratory test animals (eg. mice, rabbits, rats, guinea pigs, other rodents), and companion animals (eg. dogs, cats). In a typical embodiment, the subject is a human. Similarly, the term "individual" includes any mammal, such as humans, non-human primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys, goats), laboratory test animals (eg. mice, rabbits, rats, guinea pigs, other rodents), and companion animals (eg. dogs, cats). In a typical embodiment, the individual is a human.

[061 ] The term " stillbirth" herein refers to death of a fetus before or during birth at more than or equal to about 20 weeks gestation or of more than or equal to about 400 g birthweight (Perinatal Society of Australia and New Zealand Clinical Practice Guideline for Perinatal Mortality; Second Edition, version 2.2, April 2009). [062] The term "post-dated" refers to pregnancies beyond the expected gestation time. In the context of this disclosure, when referring to human pregnancy, the term "post-dated" is used to indicate a pregnancy greater than 40 completed weeks of gestation. A post-dated pregnancy may also be referred to as a post-term pregnancy.

[063] The term "oxidative stress", as used herein, may be used interchangeably with the term "oxidative damage" and refers to an imbalance between reactive oxygen species and antioxidant mechanisms and damage caused by such imbalance. Non-limiting examples of oxidative damage include the oxidation of nucleic acids, such as DNA or RNA, which may be detected in a sample by, for example, analysis or measurement of 8-hydroxy-deoxyguanosine or 8-hydroxyguanosine (80HdG or 8HOG), and oxidation of lipids, which may be detected in a sample by, for example, measurement of alkenals such as 4-hydroxynonenal (4HNE). In particular, herein the term "placental oxidative stress" refers to oxidative stress occurring in the placenta. Whilst not wishing to be bound by theory, the inventors propose that increased oxidative stress in the placenta is related to ageing of the placenta and may result in deficiencies in the placenta's ability to adequately sustain a fetus. The inventors hypothesise that oxidative stress in the placenta may therefore be associated with increased risk of stillbirth.

[064] The term "biomarker" as used herein refers to a naturally occurring, biologically functional compound or molecule, such as a gene, nucleic acid sequence, protein or fragment thereof, or other metabolite that has predictive value for the diagnosis of tissue aging in a variety of body tissue, such as placenta, skin, kidney and brain, for the diagnosis of pre-eclampsia, for the diagnosis of intra-uterine growth restriction, and for the diagnosis and assessment of risk of post-dated pregnancy and stillbirth. Determination of the level or activity of a marker in a biological sample may comprise the detection and quantitation of the marker itself or of a precursor, derivative or metabolite thereof. The terms "biomarker" and "marker" may be used interchangeably herein.

[065] In the context of the present disclosure, the biomarker may be an RNA or other nucleic acid sequence. In embodiments in which placental tissue aging, and related conditions and effects are assessed, the mRNA may be derived from the placenta and measured in maternal blood. Also in accordance with embodiments of the present disclosure is the determination of expression of precursors, derivatives, variants, analogues and functional fragments of biomarkers identified herein. A variant of a biomarker includes a molecule that exhibits at least some of the functional activity of the biomarker of which it is a variant.

[066] In the context of the present disclosure, reference to an increase in expression of a marker in a given sample means an increase in the level of expression of the marker in question in the sample, typically when compared to the expression levels in one or more control or reference samples or when compared with a baseline measurement of the sample in the same subject. Control or reference samples may be used herein to indicate samples obtained from one or more individuals with no signs of oxidative stress or tissue ageing and/or, in the case of pregnant individuals, not at risk of post-dated pregnancy or stillbirth.

[067] In one aspect the present disclosure provides a method for detecting or determining aging in body tissue, the method comprising measuring one or more markers of aldehyde oxidase 1 (AOXl) expression or activity in a biological sample obtained from a subject, wherein the level of AOXl expression or activity, as determined by the level(s) of the one or more markers, is indicative of aging in the tissue.

[068] A further aspect of the present disclosure provides a method for detecting placental aging, the method comprising obtaining a biological sample from a pregnant subject and measuring one or more markers of placental AOXl expression or activity in the biological sample, wherein the level of AOXl expression or activity or of the one or more markers thereof is indicative of placental aging. A further aspect provides a method for predicting the risk of stillbirth in a pregnancy, the method comprising obtaining a biological sample from a pregnant subject and measuring one or more markers of placental AOXl expression or activity in the biological sample, wherein the level of AOXl expression or activity, or of the one or more markers thereof, is indicative of risk of stillbirth.

[069] The inventors' novel findings as disclosed and exemplified herein provide, in some embodiments, an accurate, cost effective, rapid methodology for predicting the risk of post-dated pregnancy or stillbirth. Thus, in exemplary embodiments, disclosed herein is a simple biochemical test that facilitates the prediction of risk of stillbirth and the identification of therapeutic preventative or prophylactic regimes for at risk pregnancies. [070] The inventors' novel findings as disclosed and exemplified herein also provide, in some embodiments, accurate, cost effective, rapid methodologies for detecting or determining tissue aging and for detecting or determining tissue oxidative damage, in a range of body tissues, including for example placental, skin, kidney and brain tissue.

[071] The inventors demonstrate herein that between 38 and 41 weeks of gestation dramatic changes occur in the biochemistry and physiology of the placenta. In particular there is increased oxidative damage to DNA and lipid, change in position of lysosomes which accumulate at the perinuclear and basal surface of the syncytiotrophoblast, the formation of larger autophagosomes which are associated with oxidised lipid and there is increased expression of the enzyme AOXl. The same changes are observed in placentas associated with stillbirth. Similar changes in oxidation of lipid, localisation of lysosomes and size of autophagosomes occur in placental explants deprived of growth factors and these changes can be blocked by inhibition of AOXl.

[072] The inventors also demonstrate herein that the physiological function of the placenta at 41 weeks shows evidence of decline that has many features of aging in other tissues. The known exponential increase in unexplained intrauterine death that occurs after 38 weeks of gestation may therefore be a consequence of aging of the placenta and decreasing ability to function adequately to supply the increasing needs of the growing fetus. This knowledge may impact on obstetric practice to ensure infants are born before the placenta ages to the point of critical failure.

[073] As has been noted hereinbefore, the risk of stillbirth increases with gestational age, and in particular rises exponentially as gestation progresses beyond 38 weeks. Similarly, signs of oxidative damage increase as the placenta ages. However those skilled in the art will recognise that signs of placental aging, and therefor of oxidative damage, may appear at different times, and progress at different rates, in different individuals. Thus, one cannot prescribe a specific gestational age at which, or from which, methods of the present invention should be initiated, nor the number of times during pregnancy that a method should be employed. For example, a method of the present disclosure may be carried out from approximately 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks or 41 weeks gestation in the case of a human subject. The method may be carried out one or more times during pregnancy, for example every week or every two weeks. The frequency of testing and monitoring using methods disclosed herein may increase as gestational age increases. The appropriate timing for initiation of testing and monitoring in accordance with the present disclosure, and the appropriate frequency, can be determined on a case to case basis by the skilled addressee without undue burden or the need for further invention.

[074] The inventors also demonstrate herein that their findings extend to tissue other than placental tissue, providing evidence that AOX1 causes oxidative damage in the kidney.

[075] Without wishing to be bound by theory, the inventors suggest that increased AOX1 expression or activity in tissue, such as placenta, skin, kidney or brain, leads to oxidative damage and oxidative stress in the tissue and contributes to tissue aging (Figure 1, in the context of the placenta). In the case of placental tissue, aging of the placenta may result in lack of delivery of appropriate nutrients to the fetus, causing intra-uterine growth restriction and/or stillbirth. The inventors have shown that indicators of placental oxidative stress, such as placental aldehyde oxidase, can be measured in maternal blood providing a biomarker of placental integrity that may be assayed non-invasively, with minimal risk to the fetus. Accordingly, disclosed herein are suitable methods for assaying the risk of stillbirth in a pregnancy using biomarkers of placental oxidative stress, such as AOX1 expression. The inventors also demonstrate that such oxidative stress may be prevented with AOX1 inhibition.

[076] The inventors also note that indicators of placental aging, such as oxidative damage and lysosomal localisation, are shared with other pathologies such as Alzheimer's disease, and suggest that aging and oxidative stress in general may be associated with AOX1 and may also be delayed or prevented with AOX1 inhibition.

[077] In particular embodiments of the present disclosure, the methods comprise measurement of placental AOX1 mRNA in maternal blood. Additional placental oxidative stress biomarkers, as surrogates for AOX1 expression or activity, include, but are not limited to: lipid peroxidation products such as 4-hydroxynonenal (4HNE) and other hydroxyalkenals, which are indicative of oxidative damage to lipids; 8-hydroxyguanosine (80HdG) and 8-hydroxy guanosine (8HOG) which are indicative of oxidative damage to nucleic acids; and other markers indicative of oxidative stress, such as malondialdehyde (MDA), isoprostanes, oxysterols or increased protein carbonyl concentration.

[078] Embodiments of the disclosure comprise methods for detecting and determining risk of post-dated pregnancy and stillbirth, wherein an increase in AOXl mRNA or other marker of AOXl expression or activity above a baseline (as typically determined by one or more reference values as described hereinbefore) is indicative of placental oxidative stress and increased risk of post-dated pregnancy or stillbirth. As stillbirth can occur at any stage of pregnancy (by definition more than 20 weeks gestation or 400g in humans), methods disclosed herein contemplate monitoring the presence and level of oxidative stress markers disclosed herein in total maternal blood at various time points through a pregnancy. Such markers may be detected and measured from, in a human for example, 20 weeks gestation and at 1, 2, 3 or 4 week intervals over the course of the pregnancy. An increase in AOXl mRNA or marker of AOXl expression or activity from baseline will be indicative of increased risk of post-dated pregnancy or stillbirth.

[079] Biochemical tests used to determine biomarker levels in accordance with embodiments disclosed herein may be carried out utilising any means known in the art and the present disclosure is not limited by reference to the means by which the biomarker levels are determined. Determination of biomarker levels may comprise detection and/or quantitation and the methods and techniques available for such determination are well known to those skilled in the art. Typically, if the biomarker is an mRNA, an assay of the present disclosure will be quantitative real-time PCR. Other methods of detecting biomarkers of the present specification include but are not limited to PCR, DNA array, microarray, ligase chain reaction, oligonucleotide ligation assay, cDNA microarray, next generation sequencing, northern blotting, in situ hybridisation and further statistical analysis to determine differential expression in an individual.

[080] Suitable methods and techniques for measuring protein biomarkers may also include, but are not limited to, the use of spectral analysis, column chromatography, gel electrophoresis, mass spectroscopy and identification of protein spots, enzyme-linked immunosorbent assay (ELIS A), Western blot, image acquisition and analysis (such as magnetic resonance imaging (MRI) spectroscopy and single photon emission computed tomography (SPECT)), immuno staining, HPLC, LC/MA. Biochemical tests used to determine biomarker levels in accordance with embodiments disclosed herein may be employed in any suitable environment or setting, such as a hospital, clinic, surgical or medical practice, or pathology laboratory.

[081] Suitable biochemical tests may be incorporated into one or more devices capable of analysing the desired biomarkers to thereby allow a degree, or complete, automation of the testing process. Suitable devices are typically capable of receiving a biological sample, analysing one or more biomarker levels in said sample and providing data on said biomarker level(s) in real time thus facilitating bench-to-bedside and point-of-care analysis, diagnosis, risk assessment and/or treatment. It should be understood that reference to determining the level of expression or level of a marker is intended as a reference to the use of any suitable technique that will provide information in relation to the level of expression or level of the desired marker in the relevant tissue of the subject. Accordingly, these techniques include both in vivo techniques such as MRI imaging with phosphorous coils, as well as in vitro techniques that are applied to a biological sample extracted from the subject. Other oxidative stress markers, such as 4HNE, 80HdG or 8HOG may be detected by methods well known in the art, such as by fluorescent detection, immunohistochemical or chromatographic methods such as HPLC.

[082] Biological samples obtained from an individual to determine oxidative stress marker levels may be derived from any suitable body fluid or tissue. Typically, in methods of the disclosure, the sample may comprise blood (such as erythrocytes, leukocytes, whole blood, blood plasma or blood serum), urine, amniotic fluid, saliva or tissue. If the subject is pregnant, the biological sample may be of maternal, placental or fetal origin. In an embodiment, the biological sample is maternal total blood.

[083] It is known in the art that fetal cells or nucleic acids may be present in maternal blood. The biomarkers measured in the methods of this disclosure may therefore be measured in samples such as free DNA or free RNA, placental cells, fragments of placental cells, exosomes or microparticles that are released from the placenta into the maternal circulation.

[084] If increased AOX1 mRNA or biomarker levels are detected prior to naturally occurring labour a physician may consider available options to reduce stillbirth risk. Such options include, for example, induction of labour or caesarean section if the fetus is at a stage where it is likely to be viable when born. If increased AOX1 mRNA or biomarker levels are detected at an age where the infant would be considered pre-term, options may be considered to delay placental ageing or prolong the gestation. As disclosed and exemplified herein, options may include, for example, treating the pregnant mother with an antioxidant treatment or one or more AOX1 inhibitors. As further discussed hereinbelow, examples of appropriate AOX1 inhibitors include raloxifene or agonists of G protein-coupled estrogen receptor 1 (GPER1), such as G-l.

[085] Embodiments of the disclosure provide that markers of AOX 1 expression or activity may be determined or measured in combination with one or more clinical assessments as further indicators of tissue aging. For example, in a pregnant subject, clinical assessments such as altered fetal movements, measures of fetal growth or altered Doppler studies of the placental arteries, placental veins or the fetal cerebral artery may be used in combination with markers of AOX1 expression or activity to assessing placental aging, risk of stillbirth, need for intervention to prevent stillbirth and placental oxidative stress. Clinical assessments appropriate to the context may readily be determined by a person skilled in the art.

[086] The present disclosure also provides kits suitable for use in accordance with the methods of the disclosure. Such kits include, for example, prognostic kits for assaying biological samples comprising reagents for detecting expression levels of discriminatory biomarkers disclosed herein (such as AOX1 mRNA, 4HNE, 8HOG and 80HdG). Kits according to the present disclosure may also include other components required to conduct the methods of the present disclosure, such as buffers and/or diluents. The kits typically include containers for housing the various components and instructions for using the kit components in the methods of the present disclosure.

[087] The present disclosure also contemplates the administration of therapeutic agents to arrest oxidative damage or tissue aging. In some embodiments, suitable therapeutic agents may be administered during pregnancy in the case of placental oxidative damage or placental aging in a pregnancy identified at high risk of stillbirth, including when extreme prematurity precludes delivery. Without wishing to be bound by theory, the inventors suggest that the placenta may provide a tractable model of aging in a human tissue that uniquely ages in a 9 month period of time. Herein, the inventors demonstrate changes in placental oxidative stress markers and lysosomal positioning that are similar to changes in ageing diseases, such as Alzheimer's disease and Huntington's disease (see Examples). Similarities in oxidative damage and lysosymal distribution between placental aging and aging in neurodegenerative diseases suggest that there may be a similar mechanism of aging in these conditions. It is therefore contemplated in the present disclosure that ageing due to oxidative stress and diseases and conditions in which oxidative damage is pathological may be prevented, delayed or treated with administration of AOX1 inhibitors, such as GPERl agonists or raloxifene, and other inhibitors of AOX1 or GPERl agonists as contemplated herein.

[088] Accordingly, in other embodiments, suitable therapeutic agents may be administered to prevent, delay, slow, retard or treat aging and oxidative stress or damage in skin, kidney or brain tissue. Oxidative stress and damage in such tissues may be merely the result of the aging process, or may be caused, accelerated or exacerbated by a range of other factors. By way of example only, oxidative damage and aging of the skin may be associated with prolonged exposure to the sun or sunburn, oxidative damage and aging of the kidney may be associated with acute or chronic kidney injury or disease, smoking and drug abuse, hypertension, dyslipidemia, obesity and inflammatory disorders, and oxidative damage and aging of the brain may be associated with brain injury or trauma or a neurodegenerative condition such as memory loss, Alzheimer's disease and Parkinson's disease.

[089] According to methods of the present disclosure, therapeutic agents for use in accordance with the present disclosure include, for example, inhibitors of AOX1, including but not limited to raloxifene and G protein-coupled receptor 1 (GPERl) agonists. For example, in embodiments of the disclosure, agonists of GPERl may be used to reduce or prevent oxidative stress in the placenta. One particularly suitable agonist of GPERl for use in accordance with the present disclosure is G-l, (l-[4-(6-bromobenzo[l,3] dioxol-5yl)-3a,4,5,9b-tetrahydro-3H- cyclopenta[c]quinolin-8-yl]-ethanone). G-l is a cell-permeable, nonsteroidal, dihydroquinoline compound that acts as a high-affinity GPER agonist.

[090] However those skilled in the art will appreciate that the scope of the present disclosure is not so limited, and any other AOX1 inhibitor or an agent capable of activating, directly or indirectly, the GPERl receptor or its downstream pathways may be employed. Other inhibitors of AOX1 also suitable for use in methods of the disclosure may, for example, include tamoxifen, estradiol, ethinyl estradiol, phenothiazines, tricyclic antidepressants, tricyclic atypical antipsychotics, dihydropyridine calcium channel blockers, loratidine, cyclobenzaprine, amodiaquine, maprotiline, ondansetron, propafenone, domperidone, quinacrine, ketoconazole, verapamil, tacrine and salmeterol. [091 ] Additional therapies that may be used in conjunction with, or in place of, the therapeutic compounds targeting GPER1 or AOX1 include antioxidant molecules, compounds or treatments, for example thiols, ascorbic acids or phenols (such as polyphenol), superoxide dismutase, catalase, peroxiredoxin, thioredoxin, glutathione, carotenes, alpha-tocopherol, resveratrol and flavonoids.

[092] For the purposes of the present disclosure compositions comprising therapeutic agents for administration may be prepared by methods well known to those skilled in the art. Accordingly, the compositions may comprise pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants. The carriers, diluents, excipients and adjuvants must be "acceptable" in terms of being compatible with other components of the composition, and not deleterious to the subject who is to receive the composition. The pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants to be used may depend on the intended route of administration of the composition and will be well known to those skilled in the art. Compositions may be administered via any convenient or suitable route, including for example, topical, oral, nasal or other mucosal route, or parenteral, such as subcutaneous, intramuscular, or intra-arterial. The route of administration will depend on a number of factors, including the tissue to which the treatment is directed. Similarly the form or vehicle in which the composition is administered will depend on a number of factors, including the tissue to which the treatment is directed. Exemplary forms may include injectable solutions or suspensions, lotions, liniments, gels, creams, ointments, foams, oils, powders and the like for topical administration, and tablets, troches, capsules, elixirs, suspensions, syrups, wafers, granules, powders, gels, pastes, solutions, suspensions, soluble sachets, caplets, lozenges, effervescent tablets, chewable tablets, multi-layer tablets, and the like for oral administration.

[093] In one exemplary embodiment for the administration of a composition for treating, preventing, slowing, retarding or delaying skin aging or oxidative damage in the skin, a suitable AOX1 inhibitor or GPER agonist as disclosed herein may be formulated into a liniment, gel, cream, ointment, foam, oil, or the like for topical application to the skin. Such a formulation may be applied directly to the skin, or may be applied by adding the formulation to a bath. The formulation may also comprise, for example, a sunscreen, vitamin E or other suitable ingredient to minimise the effect of UV radiation and/or assist with skin moisturisation and/or repair. [094] Examples of pharmaceutically acceptable carriers, diluents, excipients and adjuvants include, but are not limited to, demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly.

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

[096] The present disclosure will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.

Examples

[097] The following examples are illustrative of the disclosure and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

General methods

Ethics, Collection and Processing of Tissues

[098] This study was approved by the human research ethics committee of the Hunter New England Health Services and the University of Newcastle, NSW, Australia. Human placentas were collected after written informed consent was obtained from the patients by midwives. Placentas were collected from women at 38-39 weeks (term) gestation undergoing caesarean section for previous caesarean section and having normal vaginal delivery and women at 41 ⁺ weeks gestation undergoing caesarean section and having normal vaginal delivery and women who had stillborn infants undergoing vaginal delivery. Placentas were collected immediately after delivery and processed without further delay. Villous tissues were sampled from multiple sites and were prepared for histology and RNA extraction. For each placenta, tissues were obtained from at least 5 different regions of the placenta and 4-5 mm beneath the chorionic plate. Samples from each individual placenta were immediately frozen under liquid nitrogen and stored at -80° C until subsequent experiments. For histology experiments, tissues were fixed in2% formaldehyde for 24 hr, stored in 50% ethanol at room temperature (RT) and embedded in paraffin. To create a placental roll a 2 cm strip of chorioamniotic membrane was cut from periphery of the placenta keeping a small amount of placenta attached to the membrane. The strip was rolled around forceps leaving residual placenta at the centre of the cylindrical roll. The cylindrical roll was then cut perpendicular to the cylindrical axis to get 4 mm thick section. The tissue was then fixed in formalin and mounted in a paraffin block. Placentas from patients with infection, diabetes, pre-eclampsia, placenta praevia, intra-uterine growth restriction or abruption were excluded.

Placental Explant Culture

[099] For in vitro experiments, human term placentas (38-39 weeks of gestation) were obtained from women with normal singleton pregnancies without any symptoms of labour after an elective (a scheduled repeat) caesarean section. Placentas were collected immediately after delivery and prepared for explant culture. Villous tissues of placentas were randomly sampled from different regions of placenta and 4-5 mm beneath the chorionic plate. Tissues were washed several times with Dulbecco's phosphate-buffered saline (DPBS) under sterile condition to remove excess blood. Villous explants of ~2 mm ³ were dissected and placed into 100 X 20 mm culture dishes (30 pieces/dish) containing 25 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1% Na-pyruvate, 1% penicillin/streptomycin (100X) solution with the addition of 10% (v/v) fetal bovine serum (FBS) and were cultured in a cell culture chamber at 37 °C temperature and maintaining 95% air (20% oxygen) and 5% C0 ₂ for 24 hours. At day 2, villous explants were transferred to fresh 30 ml growth medium and incubated in a cell culture chamber for 90 minutes. After this the tissues were washed in DMEM without FBS (refer to as ' serum-free medium' or 'growth factor deficient medium'). Next 6-7 pieces of villous tissue weighing approximately 400 mg were transferred to a culture dish (60 X 15 mm) containing 6 ml serum-free medium with or without the addition of pharmacological agents, for example, raloxifene (1 nM) and GPER1 agonist Gl (1 nM), for subsequent incubation up to 24 h. At the end of 24 h some tissues were fixed in 2% formaldehyde, subjected to routine histological processing and embedded in paraffin wax, and some tissues were immediately frozen in liquid nitrogen and stored at -80 °C until subsequent experiments. For each placental explant culture, samples were also collected at time Ό (zero)' h i.e., before incubation in serum free medium, and were formalin fixed and stored frozen at -80 °C until further experiments.

Western Blotting

[0100] Western blotting was performed according to Maiti, K et al (2011, Endocrinology, 152:2448-2455). Samples of placenta (1 g) were crushed under liquid nitrogen. Aliquots of 100 mg of placental tissues were homogenised in 1 mL of lysis buffer (phosphate buffered saline (PBS), 1% Triton-X-100, 0.1 % Brij-35, 1 X protease inhibitor, 1 X phosphatase inhibitor, pH 7.4). The protein concentration of each placental extract was measured using a BCA protein determination kit and 40 μg of placental extract was separated by electrophoresis in NuPage bis- tris precast 12 well gels for 50 mins at constant 200 V. Separated proteins were then transferred to nitrocellulose membrane using a Novex transfer system for 70 mins and blocked overnight with 1% bovine serum albumin (BSA) in tris buffered saline with 0.1 % tween-20 (TBST). The membranes were then incubated with primary antibody in 1% BSA in TBST for 2 hours, then washed three times with TBST, then incubated with HRP conjugated secondary antibodies in 1% BSA in TBST for an hour. After three further washes with TBST, the immuno -reactive bands were developed in Luminata reagent and detected using an Intelligent Dark Box LAS- 3000 Imager (Fuji Photo Film, Tokyo, Japan).

Detection of4HNE in maternal plasma

[0101] For detection and quantitation of 4HNE protein adduct in maternal plasma, the inventors used an OxiSelect™ HNE Adduct Competitive ELISA Kit (Cell Biolabs Inc, STA- 838). This assay is based on the competition between HNE conjugate and the anti-HNE antibody for HNE- protein. In this assay, at first, an HNE conjugate is coated on an ELISA plate. The unknown HNE protein samples or HNE-BSA standards are then added to the HNE conjugate reabsorbed ELISA plate. After a brief incubation, an anti-HNE antibody is added, followed by a secondary antibody

(HRP conjugated). The amount of HNE protein adducts in unknown samples is estimated from a predetermined HNE-BSA standard curve.

[0102] In this assay, at first, an HNE conjugate was coated on a 96-well plate and incubate overnight at 4 °C. HNE conjugate was removed, and the wells were rinsed twice with IX PBS. Assay Diluent was added to each well and blocked for an hour at RT and then transferred to 4 °C until use. Immediately before use, Assay Diluent was removed and 50 μΐ maternal plasma samples and HNE-BSA standards were added. The plate was then incubated for 10 min at RT on an orbital shaker. The diluted anti-HNE antibody was added to each well and incubated at RT for 2 hours on an orbital shaker. The plate was then washed 3 times with IX wash buffer with thorough aspiration between each wash. The diluted HRP conjugated secondary antibody was added to all wells and incubated for an hour at RT on an orbital shaker. The strip wells were then washed a further 3 times with IX wash buffer. The substrate solution was added to each well, incubated at RT for 2-5 minutes on an orbital shaker and the changes of colour were monitored. The stop solution was added to each well to stop the enzyme reaction. The absorbance of each well was read on a microplate reader (SPECTROstar Nano, BMG LABTECH) using 450 nm as the primary wavelength. The content of HNE protein adducts in unknown samples was determined by comparison with the predetermined HNE-BSA standard curve using GraphPad Prism software. The results were graphed, and the statistical analysis was performed using GraphPad Prism software.

Immunohistochemistry

[0103] 6 μηι paraffin placental sections were deparaffinised and hydrated, then heated with tris- EDTA buffer (pH 9) in a microwave oven for antigen retrieval. The sections were blocked with 1 % BSA in TBST for an hour at room temperature. The sections were incubated with primary antibodies overnight and washed three times with TBST, before incubation with Alexa- conjugated secondary antibodies for 90 mins. The sections were mounted with antifade solution with DAPI. The fluorescent photographs for Figures 3 to 7, 9 and 11 were taken on a Nikon eclipse 90i confocal microscope. The fluorescent photographs for Figure 10 were taken on Nikon eclipse Ti fluorescence microscope.

RNA isolation and Real time PCR

[0104] Placental tissues were crushed under liquid nitrogen. Approximately 100 mg of crushed placental tissues were homogenised in 2ml of Trizol reagent by Ultra Turrax homogenizer. Total RNA was extracted from Trizol-extract by Direct-zol™ RNA MiniPrep. The RNA was treated with DNAse and purified with a RNA Clean & Concentrator™-5 kit. The RNA quality was observed by running the DNAse treated sample in agarose gel with ethidium bromide in IX TAE buffer. The purified RNA was used to make cDNA using a Superscript® III First- Strand Synthesis System kit. The cDNA was used to run real time PCR by Taqman primers for aldehyde oxidase 1 (AOX1) and Taqman gene expression master mix with an internal control 18s ribosomal RNA to quantify mRNA for AOX1. SyBr green master mix was used to quantify mRNA for G-protein coupled receptor 1 (GPER1) with respect to beta-actin as internal control using the Applied Biosystem 7500 PCR system.

[0105] GPER1 primers were 5 ' -CGTCCTGTGCACCTTC ATGT-3 ' (forward, SEQ ID NO. 1) and 5 ' - AGCTC ATCC AGGTGAGGAAGAA-3 ' (reverse, SEQ ID NO. 2). AOX1 primers were obtained from Thermo-fisher Scientific (CAT: 4331182, assay number: Hs00154079_ml). The AOX1 TaqMan assay was designed to detect an 84 bp amplicon from base 3270 of NM_001159.3 (herein SEQ ID NO. 3), at the exon 27/28 boundary.

Statistical analysis

[0106] Sample numbers are shown in the legends to individual figures. The data in Figures 3, 4, 6, 9 and 12 were analysed by using the Mann-Whitney test (two way) and results are presented as scatter plots showing the median or standard error of the mean. The data in Figures 10 and 11 were analysed using the Wilcoxon matched-pairs signed rank test. All the p-values were calculated using the Graphpad Prism software.

Example 1 - Relationship between stillbirth risk and length of gestation

[0107] To illustrate the relationship between stillbirth risk and length of gestation the inventors created a Kaplan Myer plot of the data on human gestational length in a population with relatively low levels of medical intervention from Omigbodun and Adewuyi (1997, J Natl Med Assoc, 89: 617) and combined it with the data on risk of stillbirth per 1000 continuing pregnancies from Sutan et al. (2010, J Perinatol, 30: 311-318) (Figure 2). The data illustrate that stillbirth is consistent with an aging aetiology as defined by Johnson et al. (1999, Cell, 96: 291- 302). Example 2 -DNA/RNA and lipid oxidation in placental tissue

[0108] As oxidative damage has been observed in many aging tissues, the inventors sought evidence of placental DNA/RNA oxidation as measured by 8-hydroxy-deoxyguanosine/8- hydroxyguanosine (80HdG/8HOG), as a marker of DNA/RNA oxidation. Immunohistochemistry (IHC) was performed in placentas for 80HdG/8HOG and the average intensity of 80HdG/8HOG staining in nuclei/frame demonstrated a significant increase in DNA/RNA oxidation in post-dated and stillbirth associated placentas (Figure 3).

[0109] The increase in DNA oxidation suggested free radical damage that might also lead to lipid peroxidation. Lipid peroxidation has been observed to increase in Alzheimer's disease as measured by the formation of 4-hydroxynonenal (4HNE) (Markesbery and Lovell, 1998, Neurobiol Aging, 19: 33-36). The inventors therefore performed immunohistochemistry for 4HNE in post-dated, stillbirth and term placental tissue. This revealed a marked increase in 4HNE staining in post-dated syncytiotrophoblasts that was also observed in placentas associated with stillbirth shown in Figure 4. High levels of 80HdG/8HOG and 4HNE in post-dated and stillbirth placentas suggest that placental aging may have occurred in these placentas.

[0110] The inventors also investigated 4HNE levels in maternal plasma, using a commercial ELISA kit, in cases of early term birth, late term birth (post date delivery), pre-eclampsia and intra-uterine growth restriction (IUGR). 4HNE levels were found to be significantly higher in each of the post date delivery, pre-eclampsia and IUGR cases when compared to early term (Figure 5). Post date delivery, pre-eclampsia and IUGR are all known to be associated with increased risk of stillbirth.

Example 3 - Movement and clustering of lysosomes in post-dated and stillbirth placentas

[0111] Misfolded proteins and damaged mitochondria are normally recycled in autophagosomes in a process that involves autophagosome fusion with proteolytic enzyme containing lysosomes. Accumulation of abnormal protein is thought to play a role in aging particularly in the brain, for instance the accumulation of tau and amyloid protein in Alzheimer's disease and mutant huntin in Huntington's disease. In Huntington's disease, the distribution of the lysosomes within neurones is altered with increased perinuclear accumulation of lysosomes. The inventors used a lysosomal marker, lysosome-associated membrane protein-2 (LAMP2) to analyse the distribution of lysosomes in the placenta by immunohistochemistry. This showed lysosomes positioned on the apical surface of term placental syncytiotrophoblast (Figures 6A, 6D and 6E), whereas lysosomes relocated to the perinuclear and the basal surface in post-dated and stillbirth placentas (Figures 6B, 6C, 6F and 6G).

[0112] The present data demonstrate marked accumulation of particles positive for the lysosomal marker LAMP2 in the perinuclear and basal side of the syncytiotrophoblast of post-dated placentas and placentas associated with stillbirth.

Example 4 - Larger autophagosom.es containing 4HNE occur in post-dated and stillbirth associated placentas

[0113] Inhibition of autophagosome function with failure of fusion with lysosomes leads to an increase in autophagosome size. This process leads to inhibition of overall autophagic function that is seen in Alzheimer's disease, Danon's disease, and neurodegeneration. In the present study, the inventors detected autophagosomes using immunohistochemistry with an antibody against LC3B. A significant increase in the size of autophagosomes was observed in both postdated and stillbirth associated placentas compared to term placentas (Figure 7). Dual labelled fluorescence immunostaining showed that the larger autophagosomes of post-dated and stillbirth placentas contained 4HNE, a product of lipid peroxidation (Figure 8).

[0114] Autophagy involves fusion of acidic lysosomes with the autophagosome. The present data indicate that stillbirth and post-dated placentas contain larger autophagosomes than term placentas suggesting inhibition of the autophagic process in these placentas. These data further indicate that the autophagosomes are coated with oxidised lipid in the form of 4HNE in the failure of fusion. Such disturbances in the function of autophagosomes may lead to the accumulation of abnormal protein and deterioration in the function of the syncytiotrophoblast.

Example 5 - Role of aldehyde oxidase 1 (AOX1) in placental oxidative damage

[0115] Using methods well known to those skilled in the art, the inventors sequenced RNA isolated from six term and six post-dated placentas. Analysis of RNA sequencing data showed that out of the 20111 genes expressed in placentas, aldehyde oxidase 1 (AOX1) was significantly increased in post-dated placentas compared to term placentas (Figure 9). [0116] AOXl is a molybdoflavoenzyme which oxidises a range of aldehydes, including 4HNE, into corresponding acids and peroxides . The inventors then tested the hypothesis that AOXl was involved in the generation of the increased 4HNE observed in post-dated and stillbirth associated placentas. Dual labelled fluorescence IHC showed that AOXl co-localizes to 4HNE positive particles in post-dated (Figure lOA-C) and stillbirth placentas (Figure 10D-F). Additionally realtime qPCR using the Life Technologies TaqMan assay Hs00154079_ml confirmed that postdated and stillbirth placentas expressed significantly higher mRNA for AOXl than term placentas (Figure 10G). These data support the concept that AOXl may play a role in the oxidative damage that occurs in the post-dated and stillbirth associated placentas.

Example 6 - Pharmacological inhibition of production of 4HNE using placental explant culture

[0117] The present data provide clear evidence for increased lipid oxidation, disordered lysosome-autophagosome interactions and increased AOXl expression in the post-dated and stillbirth placental syncytiotrophoblast. To determine if these events were causally linked the inventors developed a placental explant culture system using term placental tissue cultured in serum-free (growth factor deficient) medium. IHC showed that serum deprivation significantly increased production of 4HNE at 24 hr after incubation (Figure 11A and B). A significant increase in the size of autophagosomes was also found after 24 hr incubation in serum-free medium (Figure 12A and B), suggesting inhibition of autophagy at 24 h by oxidative damage, as observed in the stillbirth and post-dated placentas. mRNA for AOXl increased at 24h suggesting involvement of AOXl in the oxidative damage.

[0118] The inventors sought to determine cause and effect relationships between the development of lipid oxidation observed when placental explants were cultured in the absence of serum and AOXl. The GPER agonist G-l was used to inhibit aldehyde oxidase. The potent AOXl inhibitor, raloxifene, was also used. G-l was selected as the inventors had detected GPER expression on the apical surface of syncytiotrophoblast (Figure 11C and 11D) and the GPER1 agonist has been shown to inhibit production of 4HNE in the kidney (Lindsey et al., 2011, Hypertension, 58: 665-671). Both raloxifene and Gl inhibited the production of 4HNE in the serum starved placental explants after 24hr of treatment (Figure 11 A and B), demonstrating these compositions were able to block the oxidative damage to the lipid and also the disturbance in autophagosome size and lysosomal location (Figure 12).

Example 7 - Presence of the cell surface estrogen receptor GPERl on the apical surface of the syncytiotrophoblast

[0119] As G-l had evident effects in placental explant cultures the inventors undertook characterisation of GPERl expression in placental tissue. The expression of GPERl in a section of placenta roll detected by fluorescent immunohistochemistry showed that GPERl is expressed in placental villi (data not shown), which at higher magnification (100X), was localised to the apical surface of placental villi, whereas term amnion, chorion and decidua showed very little GPERl expression (data not shown). Fluorescence immuno staining with an antibody against LC3B showed changes in autophagosome size in placental explants after serum starvation (Figure 12).

[0120] GPERl primers were sourced from Invitrogen: GPERl Forward primer 5'- CGTCCTGTGCACCTTC ATGT-3 ' (SEQ ID NO: l) and GPERl reverse primer 5'- AGCTCATCCAGGTGAGGAAGAA-3 ' (SEQ ID NO:2). Real time PCR for GPERl showed that placental villi have significantly higher expression of GPERl than amnion, chorion and decidua (Figure 13A). Western-blot for GPERl also confirmed higher protein levels of GPERl in placental villous tissue than amnion, chorion and decidua (Figure 13B).

[0121] The presence of the cell surface estrogen receptor GPER on the syncytiotrophoblast apical membrane is a novel finding and suggests that this receptor may play a role in modulating oxidative damage within the placenta. It has been shown that urine from pregnant women carrying a fetus with post-maturity syndrome have lower estrogen reatinine ratios than women carrying normal foetuses (Rayburn et al., 1982, Obstet Gynecol, 60: 148-153). These data support the possibility that low estrogen concentrations may lead to loss of GPER mediated inhibition of AOX1 and consequently placental oxidative damage and impaired function.

Example 8— Detection ofAOXl mRNA in maternal blood

[0122] Fetal AOX1 mRNA was detected in maternal blood by methods well known to those skilled in the art. Briefly, maternal blood samples were collected in PAXgene® Blood RNA Tubes. The blood samples were stored at -30 °C until used for RNA isolation. Total RNA in 2.5 ml of total maternal blood samples was isolated using a PAXgene Blood miRNA Kit and fully automated RNA isolation system QIAcube (Qiagen). The quality of RNA from samples was checked by running the RNA samples in a bioanalyser (Agilent Technologies). The value of the ratio of 28S and 18S greater than 1 was considered as good quality RNA obtained from total blood. The total RNA was treated with RNAse free DNAse, before it was used for preparation of cDNA. The cDNA was prepared from total RNA using the Superscript® III First-Strand Synthesis System (Life technologies). The cDNA was used to quantify the mRNA for AOX1 using the Taqman gene expression system and 7500 applied biosystem PCR machine using human 18S ribosomal RNA as an internal control.

[0123] The majority of AOX1 mRNA in maternal blood is derived from the placenta and therefore maternal blood AOX1 mRNA may be used as an indicator of placental oxidative stress. The inventors measured maternal blood AOX1 mRNA in seven mothers at term (37 - 39 completed weeks of gestation) and two women whose pregnancy resulted in a stillbirth blood samples were measured at 31 and 36 weeks gestation. The average amount of AOX1 mRNA was significantly increased in maternal blood in stillbirth as compared to term pregnancies (Figure 14).

Example 9— Aldehyde oxidase-mediated aging in the kidney

[0124] Kidney tissue from a 21 year old subject and a 66 year old subject was stained for 4HNE as a marker of oxidised lipid formed by aldehyde oxidase. The results shown in Figure 15 demonstrate that aldehyde oxidase causes oxidative damage, a marker of aging, in the kidney. Thus, tissues that express aldehyde oxidase can be aged by oxidative damage.