Quantitative evaluation index for fetal growth restriction

Abstract

The purpose of the present invention is to provide an assessment and diagnosis method for predicting neurodevelopmental disorders at an early stage, determining the optimal treatment policy, and assessing the effect of intervention treatment in newborns at an early stage, by appropriately and accurately diagnosing, with high sensitivity, perinatal brain disorders (for example, neurodevelopmental disorders) in newborns associated with fetal growth restriction. According to the present invention, provided is a method for determining, at an early stage, perinatal brain disorders of newborns caused by fetal growth restriction, the method involving (a) a process for assessing the expression levels of one or more proteins selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), neuroserpin (Serpini1), ubiquitin thioesterase OTUB1 (Otub1), and ubiquitin-like modifier activating enzyme 1 (Uba1) in a body fluid sample obtained from a subject, in comparison with the expression levels in a control body fluid.

Description

[Technical Field]

[0001] The present invention relates to a biomarker for early determination, diagnosis, or assisting in the diagnosis of perinatal disorders in newborns caused by fetal growth restriction, a method for early determination or early diagnosis of perinatal disorders in newborns using the biomarker, and a method for early determination of the effectiveness of treatment for perinatal disorders using the biomarker. [Background technology]

[0002] Pregnancy is associated with a massive increase in uterine perfusion resulting from increased maternal cardiac output and trophoblast-driven modifications of the uterine spiral arteries. Failure of this normal physiological process contributes to two of the most devastating obstetric complications: fetal growth restriction (FGR) (also called intrauterine growth restriction (IUGR)) and pre-eclampsia (PET).

[0003] Fetal growth restriction affects up to 8% of all pregnancies and is associated with high perinatal mortality, long-term neurological disorders, and increased incidence of cardiovascular disease later in life, with no effective evidence-based treatments. Severe early-onset fetal growth restriction affects 1:500 pregnancies and is associated with high mortality and long-term complications in survivors. In the most severe cases, failure to reach a viable birth weight (at least 500 g) presents a stark choice: termination of pregnancy or allowing the fetus to die in utero. Small improvements in fetal growth (e.g., to a birth weight of 700 g) and gestational age at birth (e.g., from 26 to 28 weeks) are associated with major improvements in survival and morbidity.

[0004] Current pregnancy management is designed to identify women with growth-restricted fetuses. Many strategies, such as maternal serum markers and uterine artery Doppler ultrasound testing, are available that can predict which women will develop these conditions.

[0005] To date, attempts have been made to perform therapeutic interventions such as stem cell therapy to treat perinatal brain damage in newborns due to fetal growth restriction, and the effectiveness of such treatments on subsequent neurodevelopmental disorders in infants and children has been evaluated (Patent Document 1). However, currently, there are no successful therapeutic strategies for early evaluation and diagnosis of perinatal brain damage (e.g., neurodevelopmental disorders) due to fetal growth restriction. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2017 / 199976 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention overcomes the above-mentioned problems and provides an evaluation and diagnostic method for predicting neurodevelopmental disorders early by diagnosing perinatal brain disorders (e.g., neurodevelopmental disorders) in newborns associated with fetal growth restriction with high sensitivity, appropriateness, and accuracy, and further for determining an optimal treatment plan and early evaluation of the effects of intervention in newborns. The present invention also provides information that will serve as a basis for elucidating the pathology of perinatal brain disorders associated with fetal growth restriction. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, the present inventors comprehensively analyzed proteins in cerebrospinal fluid from a rat model of fetal growth restriction, and as a result, succeeded in identifying at least six biomarkers for the evaluation, diagnosis, prediction, and treatment of developmental disorders associated with fetal growth restriction, thereby completing the present invention.

[0009] That is, the present invention is as follows. [1] A method for early assessment of perinatal disorders in newborns caused by fetal growth restriction in a mammalian subject, comprising: (a) testing the expression level of one or more proteins selected from the group consisting of alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6) in a body fluid sample obtained from the subject, compared to expression levels in normal body fluids, body fluids after treatment, or body fluids known to be indicative of perinatal disorders; and (b) assessing the subject as having a perinatal disorder if the expression level shows a statistically significant difference compared to the expression level in the normal body fluid or the body fluid after treatment, or if the expression level does not show a statistically significant difference compared to the expression level in the body fluid known to be indicative of a perinatal disorder. The above method, comprising: [2] The method according to [1], wherein the protein is selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). [3] The method according to [1] or [2], wherein the subject is a human patient. [4] The method according to any one of [1] to [3], wherein the subject is assessed as having a perinatal disorder if all of the tested proteins show a significant difference in the subject's body fluid sample compared to normal body fluid. [5] The method according to any one of [1] to [4], wherein the expression level is determined by immunoassay. [6] The method according to any one of [1] to [4], wherein the expression level is determined by liquid chromatography / mass spectrometry. [7] The method according to any one of [1] to [4], wherein the expression level is determined using a protein array. [8] Use of a proteomic profile of the expression of one or more proteins selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), neuroserpin (Serpini1), ubiquitin thioesterase OTUB1 (Otub1), and ubiquitin-like modification activating enzyme 1 (Uba1) in a body fluid obtained from a mammalian subject for early diagnosis of perinatal disorders in newborns due to fetal growth restriction in the subject. [9] The use according to [8], wherein the protein is selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1).

[10] A method for early assessment of the therapeutic effect of a perinatal disorder in a newborn caused by fetal growth restriction in a mammalian subject, comprising: (a) testing the expression level of one or more proteins selected from the group consisting of alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6) in a body fluid sample obtained from a subject undergoing treatment, compared to expression levels in normal body fluids, body fluids after treatment, or body fluids known to be indicative of perinatal disorders; and (b) determining that the treatment of the perinatal disorder in the subject is effective when the expression level does not show a statistically significant difference compared to the expression level in the normal body fluid or the body fluid after treatment, or shows a statistically significant difference compared to the expression level in the body fluid known to be indicative of a perinatal disorder; The above method, comprising:

[11] The method according to

[10] , wherein the protein is selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). [Effects of the Invention]

[0010] According to the present invention, by using the biomarker of the present invention, it is possible to predict the onset of perinatal brain damage in the neonatal period and also to predict neurodevelopmental disorders in infants or children at an early stage, thereby enabling optimal diagnostic guidelines, treatment evaluation, and elucidation of the pathology in newborns. [Brief explanation of the drawings]

[0011] [Figure 1] Alpha-2-macroglobulin (A2m) was identified as a protein showing significant variation by proteomic analysis. [Figure 2] Neuroserpin (Serpini1) was identified as a protein showing significant changes through proteomic analysis. [Figure 3] Polyubiquitin-B (Ubb) was identified as a protein showing significant variations by proteomic analysis. [Figure 4] OX-2 membrane glycoprotein (Cd200) was identified as a protein showing significant variations by proteomic analysis. [Figure 5] The ubiquitin thioesterase OTUB1 (Otub1) was identified as a protein showing significant variations by proteomic analysis. [Figure 6] Ubiquitin-like modification activating enzyme 1 (Uba1 or Ube1) was identified as a protein showing significant changes by proteomic analysis. [Figure 7A] 1 shows the expression of A2m over time in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham"). [Figure 7B]The graph shows the results of measuring A2m expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")). [Figure 7C] The results of measuring serum A2m expression over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")) are shown. [Figure 8A] 1 shows the expression of Serpini1 over time in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham"). [Figure 8B] The results of measuring Serpini1 expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")) are shown. [Figure 8C] The results of measuring serum Serpini1 expression over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")) are shown. [Figure 9A] The graph shows the expression of Ubb over time in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham"). [Figure 9B] The graph shows the results of measuring Ubb expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")). [Figure 9C] The graph shows the results of measuring serum Ubb expression over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")). [Figure 10A] The expression of Cd200 over time in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") is shown. [Figure 10B]The results of measuring Cd200 expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")) are shown. [Figure 10C] The results of measuring serum Cd200 expression over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")) are shown. [Figure 11A] 1 shows the expression of Otub1 over time in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham"). [Figure 11B] The results of measuring Otub1 expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")) are shown. [Figure 12A] 1 shows the expression of Uba1 over time in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham"). [Figure 12B] The graph shows the results of measuring Uba1 expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham-operated group ("Sham"), vehicle group ("Vehicle"), MSC group ("MSC"), and Muse group ("Muse")). [Figure 13A] Indicates the location of the hippocampus in the brain. [Figure 13B] The results of observing the expression of various biomarkers by tissue staining in cells constituting the hippocampus in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") are shown. [Figure 13C] The results of observing the expression of various biomarkers by tissue staining in cells constituting the hippocampus in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") are shown. [Figure 13D] The expression intensities of various biomarkers corresponding to FIG. 13B are shown. [Figure 13E] The expression intensities of various biomarkers corresponding to FIG. 13C are shown. [Figure 14A] Shows the location of the striatum in the brain. [Figure 14B] The results of observing the expression of various biomarkers by tissue staining in each cell constituting the striatum in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") are shown. [Figure 14C] The results of observing the expression of various biomarkers by tissue staining in each cell constituting the striatum in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") are shown. [Figure 14D] The expression intensities of various biomarkers corresponding to FIG. 14B are shown. [Figure 14E] The expression intensities of various biomarkers corresponding to FIG. 14C are shown. [Figure 15A] Indicates the location of the cerebral cortex in the brain. [Figure 15B] The results of observing the expression of various biomarkers by tissue staining in each cell that makes up the cerebral cortex in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") are shown. [Figure 15C] The results of observing the expression of various biomarkers by tissue staining in each cell that makes up the cerebral cortex in a fetal growth restriction model ("FGR") and a sham-operated group ("Sham") are shown. [Figure 15D] The expression intensities of various biomarkers corresponding to FIG. 15B are shown. [Figure 15E] The expression intensities of various biomarkers corresponding to FIG. 15C are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the following, preferred embodiments of the present invention will be described in detail for the purpose of explaining the present invention. However, it will be understood by those skilled in the art that the present invention is not limited to the preferred embodiments described below, and that various modifications may be made within the scope of the present invention.

[0013] The meanings of technical and scientific terms used in the present specification and the appended claims shall be in accordance with the meanings understood by those skilled in the art. The terms constituting the present invention will be briefly explained below.

[0014] The present invention relates to a biomarker for early determination, diagnosis, or assisting in the diagnosis of perinatal brain damage caused by fetal growth restriction, a method for early determination or early diagnosis of perinatal disorders in newborns using the biomarker, and a method for early determination of the therapeutic effect of perinatal disorders using the biomarker.

[0015] 1.Target disease (1) Fetal growth restriction Fetal growth restriction (FGR) is closely related to the life and neurological prognosis of the newborn baby. Fetal growth restriction is defined as a condition in which the fetus's growth in the womb is impaired for some reason, preventing it from growing to the appropriate age for the number of weeks. Nowadays, with the development of ultrasound equipment, it is common to diagnose FGR using ultrasound examination.

[0016] In the 1970s, advances in ultrasound diagnostic equipment and the ability to statistically analyze multiple measured fetal parameters led to the publication of a formula for calculating estimated fetal weight (EFW). In 2003, the Japan Society of Ultrasound (JSUS) created the "Standardization of Ultrasound Fetal Measurement and Reference Values ​​for Japanese Women" to standardize the various formulas for estimated weight. This was officially adopted by the Japan Society of Obstetrics and Gynecology in 2005. The summary of this formula was detailed in the "Estimated Fetal Weight and Fetal Growth Curve" health guidance manual published in 2012. Until now, fetal growth curves could only be created based on birth weight, so the creation of fetal growth curves from ultrasound measurements was a groundbreaking development. In particular, in Japan, a clinical diagnosis of fetal growth restriction is commonly made when a fetal growth curve based on the "Standardization of Ultrasound Fetal Measurement and Reference Values ​​for Japanese Women" falls below -1.5 SD.

[0017] Fetal growth restriction is a heterogeneous disorder. Fetal growth restriction that begins in the second trimester is often caused by congenital infection, genetic abnormalities, or congenital malformations, and intervention has been thought to be unlikely to improve the prognosis. However, some cases of fetal growth restriction that begin in the second trimester are caused by placental insufficiency, including hypertensive disorders of pregnancy (HDP). Because fetal growth restriction due to placental insufficiency has a worse prognosis when it begins in the second trimester than in the third trimester, it has gradually been divided into early-onset and late-onset types based on the timing of onset. While definitions vary across reports, the dividing line between 32 and 34 weeks is often used to distinguish between these two types. Regarding the prognosis of early-onset fetal growth restriction, a large-scale prospective observational study (the TRUFFLE study) was reported in 2013. Between 2005 and 2010, 503 cases of early-onset fetal growth restriction delivered between 26 and 32 weeks were registered at facilities primarily in Europe. Fetal growth restriction was defined as a fetal estimated abdominal circumference below the 10th percentile and an umbilical artery blood flow pulsatility index above the 95th percentile. Of the 503 cases, 27 (5.5%) died perinatally, and 118 (24%) had severe neonatal complications (bronchopulmonary dysplasia, grade III or higher intraventricular hemorrhage, periventricular leukomalacia, neonatal sepsis, or necrotizing enterocolitis). The prognosis for early-onset fetal growth restriction was poor. Furthermore, the time from registration to delivery, perinatal death, and severe neonatal complications were correlated with the occurrence and severity of maternal HDP. Furthermore, the TRUFFLE study followed up survival and neurological outcomes at 2 years after birth. Among surviving infants, 41 (10%) of 402 who underwent neurological evaluation were found to have neurological abnormalities. In 2017, a French population-based cohort study (EPIPAGE 2 Study) reported on the prognosis of early-onset fetal growth restriction diagnosed before 28 weeks and delivered between 22 and 31 weeks. Of 3,698 newborns, 436 (11.8%) were diagnosed with fetal growth restriction before 28 weeks. The survival rate by week was 66% at 25 weeks and over 90% at 26 to 27 weeks. The study stated that the timing of diagnosis of fetal growth restriction is important in predicting neonatal prognosis.Although late-onset fetal growth restriction has a better prognosis than early-onset fetal growth restriction, the stillbirth rate is higher than in fetuses with normal development, and problems remain. In 2015, a report titled "Shining light in dark corners" was published, focusing on management methods for late-onset fetal growth restriction (quoted from http: / / www.chugaiigaku.jp / upfile / browse / browse2521.pdf).

[0018] Fetal cell development is characterized by a rapid increase in cell number during the first trimester (from early pregnancy to 16 weeks), an increase in cell number and cell enlargement during the second trimester (17-32 weeks), and an enlargement of cells with little increase in cell number during the third trimester (after 33 weeks). Fetal growth restriction is broadly classified into three types based on fetal cell development. The etiology will be discussed in the next section, and the relationship between clinical classification and causes is shown in Table 1 below. However, there are many exceptions, and the classification into Type 1 and Type 2 is now rarely used.

[0019] [Table 1]

[0020] (i) Type 1 (symmetric type) When a fetus is damaged in the early stages of pregnancy due to an abnormality in the fetus itself, such as a chromosomal abnormality, or TORCH syndrome, cell division and proliferation in the fetal organs are inhibited, resulting in hypoplasia in which the cells that make up the organs are normal in size but have a low cell count, and this is classified as Type 1. The hypoplasia type is characterized by symmetrical growth, with both the head and trunk being equally suppressed, due to the small number of fetuses; symmetrical growth retardation is called the symmetrical type. Type 1 accounts for approximately 20% of all fetal growth retardation cases.

[0021] (ii) Composite type (intermediate type) The complex type of fetal growth restriction occurs when the cells themselves become enlarged as the number of cells increases, accounting for approximately 10% of all fetal growth restriction cases. Causes include early-onset pregnancy-induced hypertension, chronic nephritis, hypertension, and placenta-umbilical factors.

[0022] (iii) Type 2 (asymmetric type) When disorders occur during the third trimester, cell division has already ceased, suppressing cell hypertrophy. When the cell count is normal but the cells themselves are small, a condition known as Type 2 malnutrition occurs. It is often caused by pathological placental abnormalities resulting from maternal diseases such as pregnancy-induced hypertension or diabetes in the late stages of pregnancy. When fetal placental circulation deteriorates, vasodilation (blood flow redistribution) occurs to protect vital organs such as the brain, heart, and adrenal glands. Maintaining cerebral blood flow takes priority, maintaining head development. However, blood flow to internal organs such as the trunk, liver, and intestines decreases, resulting in growth disorders characterized by a small abdomen and a thin, emaciated body with little subcutaneous fat, described as asymmetric. Type 2 (asymmetric type) accounts for approximately 70% of all fetal growth restriction cases. However, it should be noted that if the brain sparing effect breaks down and head development is impaired, the child will transition to a symmetrical type (quoted from http: / / www.chugaiigaku.jp / upfile / browse / browse2521.pdf).

[0023] (2) Perinatal brain injury "Perinatal brain damage" refers to brain damage that occurs during the perinatal period (in humans, from 22 weeks of gestation to less than 7 days after birth), such as brain damage associated with hypoxic-ischemic encephalopathy during parturition or systemic inflammatory response syndrome (SIRS) secondary to viral or bacterial infection. The present invention also covers the period when neurodevelopmental disorders caused by fetal growth restriction manifest as symptoms (e.g., in humans, between the ages of 2 and 3). More specifically, the present invention targets perinatal brain damage that occurs in human newborns (within 28 days of birth), infants (less than 1 year of age), and toddlers (1 to 6 years of age).

[0024] "Cerebral palsy" refers to irreversible brain damage that occurs during the brain development period (in humans, this refers to the period from the 13th day of pregnancy to the 48th day after birth). It is a non-progressive disorder, and its symptoms are primarily motor dysfunction, with most cases occurring by the age of three. Specifically, it generally refers to brain damage that occurs by the neonatal period. Causes are classified according to the time of onset of the damage: (a) prenatal causes include intrauterine infection, placental insufficiency, fetal cerebrovascular disease, and hereditary causes; (b) intranatal causes include mechanical injury during delivery, cerebral hemorrhage, anoxia, hypoxia, and cerebral circulatory disorders; and (c) postnatal causes include severe jaundice (kernicterus), intracranial infection, and cerebral hemorrhage. Classification is based on the type of paralysis. Muscle tone can include spasticity, rigidity, ataxia, athetosis (involuntary movements that occur when attempting to maintain a certain posture or perform a movement), and atonia. Paralysis can range from quadriplegia, hemiplegia, diplegia, paraplegia, diplegia, and monoplegia. Complications include intellectual disability (including learning disabilities), epileptic seizures, cranial nerve disorders, and speech disorders. Common brain lesions caused by hypoxic-ischemic encephalopathy in mature infants include cerebral cortical laminar necrosis, basal ganglia necrosis, cerebral infarction, leukomalacia, and pontine uncinate gyrus necrosis. Necrosis of the brain stem is also reported. Clinically, basal ganglia necrosis causes athetoid cerebral palsy, while cerebral infarction causes spastic quadriplegia and hemiplegia. Brain stem necrosis has a poor prognosis and often results in death during infancy. Surviving infants may experience swallowing disorders and respiratory control abnormalities.

[0025] For example, brain ultrasound, MRI, CT, EEG, and laser Doppler flowmetry can be used to obtain physiological findings of the brain. Abnormal findings using these devices can raise suspicion of perinatal brain damage. Learning and motor disorders can also be diagnosed by direct observation in subjects.

[0026] 3. Method for early determination, diagnosis, or assistance in diagnosis of perinatal disorders in newborns using the biomarkers of the present invention According to the present invention, a method for early assessment, diagnosis, or support of perinatal brain damage in newborns due to fetal growth restriction in a mammalian subject (hereinafter simply referred to as the "early diagnosis method") is characterized by the use of one or more of the six biomarkers described below. The biomarkers (proteins) used are identified by comparing the expression levels of proteins present in the body fluids of newborns suspected of having the disease with those present in normal body fluids, body fluids after treatment, or body fluids of subjects known to exhibit the disease, and identifying the presence or absence of differences or up- or down-regulated proteins. The totality of proteins present in a biological sample (e.g., body fluid, tissue, organism, or cell culture) at a given time point is generally referred to as the "proteome." Protein expression levels can be determined using immunoassays, mass spectrometry, or protein arrays.

[0027] Such expression comparisons may be used to study global changes in protein expression in a sample (also called "proteomics" or "expression proteomics"). Proteomics generally involves the steps of: (1) separating individual proteins in a sample by two-dimensional gel electrophoresis; (2) identifying the individual proteins recovered from the gel, for example, by liquid chromatography, mass spectrometry, or N-terminal sequencing; and (3) analyzing the data using bioinformatics. Proteomics methods can be a useful complement to other gene expression profiling methods.

[0028] The present invention primarily involves proteomic analysis of body fluids. The "body fluids" used include, but are not limited to, cerebrospinal fluid (CSF), umbilical cord blood, cervical-vaginal fluid (CVF), amniotic fluid, blood, serum, plasma, urine, breast milk, mucus, saliva, and sweat. In the present invention, to diagnose perinatal brain damage associated with fetal growth restriction, which poses a risk for neurodevelopmental disorders, CSF is preferred as the body fluid. This is because information on biomolecules circulating in the brain due to the inflow and outflow of CSF sensitively reflects the state and changes in brain tissue. Furthermore, serum, which is the destination of CSF outflow, is useful because it can be collected noninvasively and easily.

[0029] In comparative analysis, it is of course important to treat normal and biological samples accurately and in the same way to accurately represent the relative expression levels or amounts of proteins and obtain accurate results. The required amount of total protein depends on the analytical technique used and can be easily determined by those skilled in the art. Proteins present in biological samples are generally separated by two-dimensional gel electrophoresis according to their pI and molecular weight. Proteins are first separated by their charge using isoelectric focusing (one-dimensional gel electrophoresis). This step may be performed, for example, using commercially available immobilized pH gradient (IPG) stripes. The second dimension may be conventional SDS-PAGE analysis, in which concentrated IPG strips are used as samples. After separation, proteins can be visualized using conventional dyes such as Coomassie blue or silver stain and imaged using known techniques and equipment, for example, a Bio-Rad GS800 densitometer and PDQUEST software. Individual spots are then excised from the gel, destained, and digested with trypsin. The peptide mixture may be analyzed by mass spectrometry (MS). Alternatively, the peptides may be separated, for example, by capillary high performance liquid chromatography (HPLC) and analyzed by MS, either separately or in combination.

[0030] A mass spectrometer consists of an ion source, a mass analyzer, an ion detector, and a data collection unit. Fragmented peptides are ionized in the ion source. The ionized peptides are then separated according to their mass-to-charge ratio in the mass analyzer, and different ions are detected. In particular, since the development of matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) methods, mass spectrometry has been widely used in protein analysis. Examples include MALDI-TOF and quadrupole-TOF, or an ion trap mass analyzer coupled with ESI.

[0031] Protein arrays are formed by immobilizing proteins on solid surfaces, such as glass, silicon, microwells, nitrocellulose, PVDF membranes, and microbeads, using a variety of covalent and non-covalent attachment chemistries well known in the art. The solid support must be chemically stable before and after the coupling procedure, allow for good spot morphology, exhibit negligible nonspecific binding, not affect the background of the detection system, and be compatible with different detection systems.

[0032] The diagnostic methods of the present invention may be implemented in the form of various immunoassay formats, which are well known in the art. There are two main types of immunoassays: homogeneous and heterogeneous. In homogeneous immunoassays, the immune reaction between antigen and antibody and its detection are carried out in a homogeneous reaction. Heterogeneous immunoassays include at least one separation step to separate the reaction product from unreacted reagents.

[0033] The six proteins used in the present invention are proteins (or peptides) discovered from a total of 601 species based on the comprehensive proteomics analysis described above. Specifically, they are alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Information on the amino acid sequences of these proteins is publicly available from publicly available databases (e.g., UniProt; https: / / www.uniprot.org / ).

[0034] The present invention's early diagnosis of perinatal brain damage in newborns utilizes at least one, preferably at least two, three, four, or five, or all, of the above proteins. As shown in the Examples below, four of the six proteins (i.e., alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpinil)) are present in cerebrospinal fluid and are significantly and chronically altered by fetal growth restriction. Furthermore, it has been shown that the administration of pluripotent stem cells (Muse cells) improves the altered expression of these proteins at an early stage. Furthermore, at least three of these four proteins (i.e., alpha-2-macroglobulin (A2m), polyubiquitin-B (Ubb), and neuroserpin (Serpini1)) were also identified in the serum of newborns, and significant changes in expression were observed in association with fetal growth restriction, and these changes were improved by administration of pluripotent stem cells (Muse cells).

[0035] The variation in the expression levels of the six or four proteins in a body fluid sample is measured by comparing them with the expression levels in a normal body fluid, a body fluid after treatment (with a drug, pluripotent stem cells, etc.), or a body fluid known to indicate perinatal disorders. When the body fluid used as the comparison standard is a normal body fluid or a body fluid after treatment, the variation may be an increase or decrease in expression level of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more. Furthermore, when the bodily fluid used as a comparison standard is a bodily fluid known to indicate perinatal disorders, the difference from it is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less of the expression level of the reference bodily fluid, or preferably 0%.

[0036] Another aspect of the present invention provides a method for early assessment of the therapeutic effect of a perinatal disorder in a mammalian subject, which comprises detecting at least one, preferably at least two, three, four, or five, or all of the above-mentioned proteins: alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Using all of these, the expression level of the protein in a body fluid sample obtained from the subject under treatment is tested in comparison with the expression level in a normal body fluid, a body fluid after treatment, or a body fluid known to be indicative of a perinatal disorder; and if the expression level does not show a statistically significant difference compared to the expression level in the normal body fluid or the body fluid after treatment, or shows a statistically significant difference compared to the expression level in the body fluid known to be indicative of a perinatal disorder, it can be determined that the treatment of the perinatal disorder in the subject is effective.

[0037] The variation in the expression level of the above protein in a body fluid sample is determined by comparing it with the expression level in a normal body fluid, a body fluid after treatment (with a drug, pluripotent stem cells, etc.), or a body fluid known to indicate perinatal disorders. When the body fluid used as the comparison standard is a normal body fluid or a body fluid after treatment, the difference from the expression level of the reference body fluid is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less, or preferably 0%, of the expression level of the reference body fluid. Furthermore, when the body fluid used as the comparison standard is a body fluid known to indicate perinatal disorders, the fluctuation may be an increase or decrease in expression level of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more based on the body fluid.

[0038] The present invention can provide a method for detecting mild perinatal brain damage that is difficult to detect using current diagnostic methods, thereby enabling early diagnosis of brain damage (e.g., neurodevelopmental disorders) in infants and children after development. According to the present invention, brain damage that occurs after development, which has been overlooked until now, can be diagnosed in the neonatal period, making it possible to detect perinatal brain damage that has not been diagnosed until now.

[0039] As described in the Examples below, the characteristics of six proteins obtained by comprehensive analysis of proteins in cerebrospinal fluid from a rat model of fetal growth restriction and associated with markers of perinatal brain damage due to fetal growth restriction or therapeutic markers are outlined below.

[0040] (a) A2m (α-2-macroglobulin) Characteristics: A type of acute phase response protein. Increased in response to cell injury. In the brain, it increases due to breakdown of the blood-brain barrier (BBB) ​​or injury to nerve cells. Results: A significant increase was confirmed in serum and cerebrospinal fluid on both days 5 and 14 after birth. Expression suppression by Muse cells was also confirmed on both days 5 and 14 after birth. While the suppressive effect of Muse cells could not be confirmed in cerebrospinal fluid on day 5 after birth, it was confirmed in serum. This is thought to be because fetal growth restriction induces increased expression of A2m in organs other than the brain (such as the lungs), and Muse cells act on those organs earlier than on the brain.

[0041] (b) Serpini1 (neuroserpin) Characteristics: Neuroserpin is a type of serine protease inhibitor that is expressed in large quantities in the brain. It inhibits proteolytic enzymes, thereby suppressing protein breakdown, and also plays a role in controlling inflammation. It is important for the normal function of nerve cells, and is said to control memory and emotions. It has also been reported to have neuroprotective effects. Malfunction of this protein can lead to dementia. Results: Significant increases were confirmed in serum and cerebrospinal fluid on both days 5 and 14 after birth. Similar to cerebrospinal fluid, serum samples showed a greater increase on day 14 after birth. Furthermore, compared to cerebrospinal fluid, the increase was greater, as it was barely detectable in the control. Expression suppression by Muse cells and mesenchymal stem cells (MSCs) was also confirmed on both days 5 and 14 after birth. The suppressive effect was more pronounced in the Muse cell group and is thought to persist over the long term.

[0042] (c) Cd200 (OX-2 membrane glycoprotein) Characteristics: A protein that mainly suppresses immune system cells and regulates inflammation. In the brain, it is highly expressed in neurons. Signaling from Cd200 / Cd200R causes the secretion of anti-inflammatory cytokines, which suppress inflammation. Results: A significant increase was confirmed 5 days after birth, but the inhibitory effect of stem cells only showed a tendency.

[0043] (d) Ubb (polyubiquitin-B) Characteristics: This protein functions as a label attached to remove unnecessary or abnormal proteins. Increased expression of this protein indicates an increase in unnecessary proteins. Abnormal expression fluctuations can also cause neurodegenerative diseases. Results: A significant increase was confirmed on both days 5 and 14 after birth. Expression was also suppressed by Muse cells on both days 5 and 14 after birth. The effect was faster with Muse cells than with MSCs.

[0044] (e) Uba1 (ubiquitin-like modification activating enzyme 1) Characteristics: A type of ubiquitin-activating enzyme, it is a protein that activates ubiquitin used in proteolysis so that it can be used in the next proteolysis. Like Ubb, it is an important molecule for protein quality control. Uba1 is sometimes referred to as "Ube1."

[0045] (f) Otub1 (ubiquitin thioesterase OTUB1) Characteristics: Deubiquitinating enzyme. A protein that removes ubiquitin from ubiquitinated proteins and chromatin. Like Ubb, it is an important molecule for protein quality control.

[0046] Another aspect of the present invention provides the use of one or more of any proteins selected from the group consisting of alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6) in the production of a proteomic (or protein) profile of a body fluid in a mammalian subject for the early diagnosis of perinatal disorders in newborns due to fetal growth restriction.

[0047] In some embodiments, the proteomic profile includes information on the expression levels of at least two, at least three, at least four, at least five, or all of the above proteins, in any combination.

[0048] In one embodiment, the proteomic profile contains information on the expression levels of the above proteins, and if one or more of the tested proteins show a statistically significant difference compared to their expression levels in normal body fluids or body fluids after treatment, or do not show a statistically significant difference compared to their expression levels in said body fluids known to be indicative of a perinatal disorder, the subject is diagnosed with a perinatal disorder.

[0049] In the present invention, when the body fluid used as a comparison standard is a body fluid after treatment, the "treatment" may be, but is not limited to, a treatment for perinatal brain injury (e.g., hypothermia therapy) or stem cell therapy (e.g., umbilical cord blood stem cells, Muse cells), as recognized by those skilled in the art.

[0050] "Muse (Multilineage-differentiating Stress Enduring) cells" can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov. 20, 2013 (Epub) (published on Jan. 17, 2014)), and skin tissues such as dermal connective tissue. They are also found scattered in the connective tissues of various organs. These cells possess properties of both pluripotent stem cells and mesenchymal stem cells and are identified, for example, by double positivity for the respective cell surface markers "SSEA-3 (Stage-specific embryonic antigen-3)" and "CD105." Therefore, Muse cells or cell populations containing Muse cells can be isolated from biological tissues, for example, using these antigen markers as indicators. Muse cells are stress-resistant and can be enriched from mesenchymal tissues or cultured mesenchymal cells by various stress stimuli. Cell fractions enriched in Muse cells by stress stimuli can also be used for the cell preparations of the present invention. Details of the isolation, identification, and characteristics of Muse cells are disclosed in International Publication No. WO 2011 / 007900. Furthermore, as reported by Wakao et al. (2011, supra), when mesenchymal cells are cultured from bone marrow, skin, etc. and used as a population of Muse cells, all SSEA-3-positive cells are CD105-positive. Therefore, when isolating Muse cells from in vivo mesenchymal tissue or cultured mesenchymal stem cells, Muse cells can be purified and used simply using SSEA-3 as an antigen marker. Note that, herein, pluripotent stem cells (Muse cells) or cell populations containing Muse cells isolated from in vivo mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker may be referred to simply as "SSEA-3-positive cells." Furthermore, herein, "non-Muse cells" refers to stem cells contained in in vivo mesenchymal tissue or cultured mesenchymal tissue, other than "SSEA-3-positive cells."According to the method described in International Publication No. WO2011 / 007900 regarding the isolation and identification of human Muse cells, a cell population obtained by removing SSEA-3 and CD105-positive cells from MSCs can be used as non-Muse cells.

[0051] Briefly, Muse cells or stem cell populations containing Muse cells can be isolated from biological tissues (e.g., mesenchymal tissues) using an antibody against the cell surface marker SSEA-3 alone or using antibodies against SSEA-3 and CD105 together. Here, "living body" refers to a mammalian living body. In the present invention, living body does not include fertilized eggs or embryos at developmental stages earlier than the blastula stage, but does include embryos at developmental stages after the blastula stage, including fetuses and blastulas. Mammals include, but are not limited to, humans, primates such as monkeys, rodents such as mice, rats, and guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparations and pharmaceutical compositions of the present invention are clearly distinguishable from embryonic stem cells (ES cells) and iPS cells in that they are directly isolated from biological tissues using markers. Furthermore, "mesenchymal tissue" refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, and umbilical cord blood, as well as tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, or adipose tissue. For example, it is preferable to collect mesenchymal tissue from a living body and isolate and use Muse cells from this tissue. Alternatively, Muse cells may be isolated from cultured mesenchymal cells such as fibroblasts or bone marrow mesenchymal stem cells using the above-mentioned isolation methods. In the cell preparations and pharmaceutical compositions of the present invention, the Muse cells used may be autologous or allogeneic to the recipient.

[0052] As described above, Muse cells or cell populations containing Muse cells can be isolated from biological tissues using, for example, SSEA-3 positivity and SSEA-3 and CD105 double positivity as indicators. However, adult human skin is known to contain various types of stem and progenitor cells. However, Muse cells are not the same as these cells. These stem and progenitor cells include skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs), melanoblasts (MBs), pericytes (PCs), endothelial progenitor cells (EPs), and adipose-derived stem cells (ADSCs). Muse cells can be isolated by detecting the absence of markers specific to these cells. More specifically, Muse cells can be separated using non-expression of at least one, for example, two, three, four, five, six, seven, eight, nine, ten, or eleven, of eleven markers selected from the group consisting of CD34 (a marker for EPs and ADSCs), CD117 (c-kit) (a marker for MBs), CD146 (a marker for PCs and ADSCs), CD271 (NGFR) (a marker for NCSCs), NG2 (a marker for PCs), vWF factor (von Willebrand factor) (a marker for EPs), Sox10 (a marker for NCSCs), Snail (a marker for SKPs), Slug (a marker for SKPs), Tyrp1 (a marker for MBs), and Dct (a marker for MBs). For example, but not limited to, separation can be performed using the non-expression of CD117 and CD146 as an indicator, and further separation can be performed using the non-expression of CD117, CD146, NG2, CD34, vWF, and CD271 as an indicator, and further separation can be performed using the non-expression of the above 11 markers as an indicator.

[0053] Furthermore, Muse cells having the above characteristics are as follows: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) Self-renewing ability In one aspect of the present invention, Muse cells used in the cell preparations and pharmaceutical compositions of the present invention have all of the above properties. Regarding (i) above, "low or no telomerase activity" refers to low or undetectable telomerase activity when detected using, for example, a TRAPEZE XL telomerase detection kit (Millipore). "Low" telomerase activity refers to telomerase activity comparable to that of somatic human fibroblasts, or telomerase activity that is one-fifth or less, preferably one-tenth or less, of that of HeLa cells. Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endodermal, mesodermal, and ectodermal) in vitro and in vivo. For example, by in vitro induction culture, they can differentiate into hepatocytes, neurons, skeletal muscle cells, smooth muscle cells, osteocytes, adipocytes, etc. Furthermore, they may also exhibit the ability to differentiate into three germ layers when transplanted into testes in vivo. Furthermore, when transplanted intravenously into the body, Muse cells have the ability to migrate and engraft in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells appropriate for the tissue. Regarding (iii) above, Muse cells proliferate at a rate of approximately 1.3 days in suspension culture. However, in suspension culture, they proliferate from a single cell, form embryoid-like cell clusters, and proliferation slows after approximately 14 days. However, when these embryoid-like cell clusters are cultured in adherent culture, cell proliferation resumes, and the cells proliferate from the cell clusters and spread. Furthermore, when transplanted into the testis, they do not become cancerous for at least six months. Regarding (iv) above, Muse cells have the ability to self-renew (self-replicate). Here, "self-renewal" refers to the process by which differentiation into three germ layers can be confirmed from cells contained in an embryoid-like cell mass obtained by culturing a single Muse cell in suspension culture, and at the same time, by bringing the cells from the embryoid-like cell mass back into suspension culture as a single cell, an embryoid-like cell mass of the next generation can be formed, from which differentiation into three germ layers and the formation of an embryoid-like cell mass in suspension culture can again be confirmed. Self-renewal can be performed by repeating one or more cycles.

[0054] Furthermore, the cell fraction containing Muse cells may be a cell fraction enriched in SSEA-3-positive and CD105-positive pluripotent stem cells that have at least one, and preferably all, of the following properties, and that is obtained by a method comprising applying an external stress stimulus to mesenchymal tissue of a living body or cultured mesenchymal cells, killing cells other than those resistant to the external stress, and recovering the surviving cells: (i) SSEA-3 positive; (ii) CD105 positive; (iii) low or absent telomerase activity; (iv) have the ability to differentiate into three germ layers; (v) does not exhibit neoplastic growth; and (vi) It has self-renewal ability.

[0055] The external stress may be any one or a combination of protease treatment, culture under low oxygen concentration, culture under low phosphate conditions, culture under low serum concentration, culture under low nutrient conditions, culture under heat shock, culture at low temperature, freezing treatment, culture in the presence of harmful substances, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical impact. For example, the protease treatment time is preferably 0.5 to 36 hours in total to impart external stress to the cells. Furthermore, the protease concentration may be any concentration used when detaching cells adhered to a culture vessel, disaggregating cell clumps into single cells, or recovering single cells from tissue. The protease is preferably a serine protease, an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a glutamic acid protease, or an N-terminal threonine protease. Furthermore, the protease is preferably trypsin, collagenase, or dispase.

[0056] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples in any way. [Example]

[0057] Example 1. Creation of a fetal growth restriction model animal and animal management Sixty 11-week-old SD rats on day 4 of pregnancy were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). They were randomly assigned to one of the following groups: a sham-operated group (Sham group, n = 15), a fetal growth restriction model group administered with balanced salt solution (Vehicle group, n = 15), a fetal growth restriction model group administered with mesenchymal stem cells (MSC group, n = 15), or a fetal growth restriction model group administered with Muse cells (Muse group, n = 15). Each group consisted of one pregnant rat per cage, housed at a room temperature of 22–24°C, humidity of 50–60%, and a 12-hour light-dark cycle with free access to food and water. On day 17 of pregnancy, pregnant rats in the vehicle, MSC, and Muse groups underwent intrauterine hypoperfusion by placing ameroid constrictors (Research Instruments SW, CA, US) on the uterine and ovarian arteries. The uterus, exposed when the ameroid constrictor was attached, was wrapped in gauze soaked in saline heated to 37°C to prevent drying and a drop in body temperature. For pregnant rats in the sham group, only the abdominal opening and uterine exposure procedures were performed on day 17 of pregnancy, as with the vehicle group, and the abdomen was closed without the attachment of an ameroid constrictor.

[0058] On day 21 of pregnancy, the birth of pups from each pregnant rat was confirmed, and female pups were randomly culled to ensure 10 pups per dam. The weight transition of all male pups was assessed from day 3 to day 14 after birth, and cerebrospinal fluid was collected from pups on days 4, 5, 7, 10, and 14 after birth, and blood was collected from pups on days 5 and 14 after birth. Furthermore, on day 4 after birth, balanced salt solution was administered via the jugular vein to the sham and vehicle groups, mesenchymal stem cells were administered to the MSC group, and Muse cells were administered to the Muse group at 1.0 × 10 4All animal experiments were performed in accordance with the "Animal Research: Reporting In Vivo Experiments guidelines for the care and use of laboratory animals" and were approved by the Nagoya University Animal Experiment Ethics Committee (approval numbers 30082 and 1107). All surgeries and sample collections were performed under isoflurane anesthesia, and every effort was made to minimize pain and suffering.

[0059] Example 2. Collection of cerebrospinal fluid To collect cerebrospinal fluid, capillaries (DRM Microcap, 1-000-0500) were purchased and sharpened by crushing the tip. The posterior fossa of a rat pup was exposed after hypnotizing it with isoflurane, and the sharpened capillary was inserted into the cisterna magna to collect cerebrospinal fluid via capillary action. The collected cerebrospinal fluid was placed in a tube containing a protease inhibitor, immediately frozen in liquid nitrogen, and stored frozen at -80°C until use.

[0060] Example 3. Collection of serum 500 μL of blood was collected from the heart of rat pups sedated with isoflurane and placed in a microtube (BD Microtainer SST) containing a coagulant and serum separator. After mixing by inversion five times, blood cells were precipitated by centrifugation at 10,000 × g, and serum was collected. Proteinase inhibitors were added to the collected serum, which was then frozen at -80°C until use.

[0061] Example 4. Comprehensive analysis of proteins in cerebrospinal fluid by proteomics The protein concentration of the collected cerebrospinal fluid was measured by the BCA method and standardized to 50 μg / 100 μL. The total protein abundance in the cerebrospinal fluid was measured by liquid chromatography / mass spectrometry (LC / MS) to obtain a protein profile. Spearman correlation coefficients were calculated between the abundance of all 601 proteins detected in the cerebrospinal fluid and body weight on day 3 of birth, and p-values ​​and false discovery rates (FDRs) were calculated based on the correlation coefficients. It has previously been confirmed that body weight on day 3 of birth correlates with birth weight. Proteins with p<0.05 and FDR<0.10 were identified: 140 on day 4 and 123 on day 5 of birth, for a total of 212 proteins (51 overlapping proteins). These 212 proteins were identified as proteins that change with fetal growth restriction. Functional analysis of these 212 proteins using Gene Ontology (GOT) revealed protein clusters that met the following criteria: p<0.05, Fold Enrichment>2, and Symbols number>3. For the 140 proteins on day 4, 333 protein clusters related to biological processes, 42 protein clusters related to cellular components, and 50 protein clusters related to molecular function were extracted. For the 123 proteins on day 5, 191 protein clusters related to biological processes, 27 protein clusters related to cellular components, and 35 protein clusters related to molecular function were extracted. Among these terms, the top-ranked terms showing particularly significant changes were those related to neural stem cell and glial cell differentiation, neurogenesis, and cell death regulation on day 4, and those related to synapse formation, neural circuit development, and behavioral function on day 5. Furthermore, protein clusters related to inflammation and immune responses, cell adhesion, and protein structure management were commonly extracted on both days 4 and 5. From the group of proteins that changed commonly on days 4 and 5 of birth, proteins that increased or decreased commonly at both time points on days 4 and 5 of birth were extracted, and 14 proteins were found.Furthermore, we investigated which of these 14 proteins were expressed in the brain using The Human Protein Atlas, and found six proteins: alpha-2-macroglobulin (A2m) (Figure 1), neuroserpin (Serpini1) (Figure 2), polyubiquitin-B (Ubb) (Figure 3), OX-2 membrane glycoprotein (Cd200) (Figure 4), ubiquitin thioesterase OTUB1 (Otub1) (Figure 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (Figure 6).

[0062] Example 5. Protein quantification by polyacrylamide gel electrophoresis and Western blotting CSF and serum proteins were mixed with sample denaturing buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 4% w / v sodium dodecyl sulfate [SDS], 0.001% w / v bromophenol blue, and 10% mercaptoethanol) and heated at 95°C for 5 min to denature the protein structure. Five μg of protein per lane from each sample was loaded onto a 10% SDS-polyacrylamide gel and electrophoresed at 80 V and 10°C for 120 min. The separated proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore, MA, USA) at 400 mA for 1 h at room temperature. After blocking the proteins on the membrane with 5% skim milk dissolved in Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 (TBS-T), the primary antibodies were rabbit anti-albumin (Proteintech: 16475-1-AP, 1:5000), rabbit anti-A2m (Abcam: ab58703, 1:500), rabbit anti-Cd200 (Proteintech: 14057-1-AP, 1:200), rabbit anti-Ube1 (Uba1) (Proteintech: 15912-1-AP, 1:400), rabbit anti-Otub1 (Abcam: ab101471, 1:500), rabbit anti-neuroserpin (Abcam: ab33077, 1:200), or rabbit anti-Ubb (Stressmarq) antibodies. The primary antibody-incubated membrane was incubated overnight at 4°C with a secondary antibody, horseradish peroxidase goat anti-rabbit IgG antibody (Rockland: 611-603-122, 1:10,000 for albumin and 1:1,000 for others) at room temperature for 1 hour. Between each step, the membrane was washed with TBS-T. Secondary antibody binding was visualized and detected by chemiluminescence using Immobilon Westem Chemiluminescent HRP Substrate (Merck Millipore). To quantify the detected signal, images were scanned using an ImageQuant LAS 4000 (GE Healthcare, IL, US) and analyzed using Image J software.Polyacrylamide gel electrophoresis confirmed the appearance of bands at 66 kDa for rat albumin, 163 kDa for A2m, 46 kDa for neuroserpin, 34 kDa for Ubb, 41 kDa for CD200, 31 kDa for OTUB1, and 117 kDa for UBE1. The density of each detected band was quantified by background subtraction, and the quantitative values ​​for A2m, neuroserpin, Ubb, Cd200, OTUB1, and UBE1 were corrected for the quantitative value of albumin in each sample (Figures 7A-12A).

[0063] The abundance of each protein expressed in various fetal growth restriction model groups (sham group, vehicle group, MSC group, and Muse group) was measured and the results are shown in Figures 7B and C, 8B and C, 9B and C, 10B and C, 11B, and 12B). Figures 7B, 8B, 9B, 10B, 11B, and 12B show the abundance of each protein in cerebrospinal fluid (days 5, 7, 10, and 14 after birth), while Figures 7C, 8C, 9C, and 10C show the abundance of each protein in serum (days 5 and 14 after birth).

[0064] (a) A2m (α-2-macroglobulin) A significant increase in A2m expression was confirmed in serum and cerebrospinal fluid on both postnatal days 5 and 14. Furthermore, suppression of expression by Muse cells was confirmed on both postnatal days 5 and 14 (Fig. 7B and C). Although the suppressive effect of Muse cells was not confirmed in cerebrospinal fluid on postnatal day 5 (Fig. 7B), it was confirmed in serum. This is thought to be because fetal growth restriction induces increased expression of A2m in organs other than the brain (e.g., lungs), and Muse cells act on these organs earlier than on the brain.

[0065] (b) Serpini1 (neuroserpin) A significant increase was confirmed in serum and cerebrospinal fluid on both days 5 and 14 after birth. Similar to the cerebrospinal fluid (Fig. 8B), the serum sample (Fig. 8C) showed a greater increase on day 14 after birth. Furthermore, the increase was greater than in the cerebrospinal fluid, as it was barely detectable in the control. Furthermore, suppression of expression by Muse cells and mesenchymal stem cells (MSCs) was confirmed on both days 5 and 14 after birth (Fig. 8C). The suppressive effect was more pronounced in the Muse cell group and is thought to persist over the long term.

[0066] (c) Ubb (polyubiquitin-B) A significant increase was confirmed on both days 5 and 14 after birth. Furthermore, suppression of expression by Muse cells was confirmed on both days 5 and 14 after birth. The effect was faster with Muse cells than with MSCs (Figure 1). 9C ).

[0067] (d) Cd200 (OX-2 membrane glycoprotein) Although a significant increase was confirmed on postnatal day 5, the inhibitory effect of stem cells was only a trend (Fig. 10B and C).

[0068] (e) Otub1 (ubiquitin thioesterase OTUB1) In the cerebrospinal fluid, Muse cells and MSCs suppressed expression on both postnatal days 5 and 14. The effect was faster with Muse cells than with MSCs (Figure 11B).

[0069] (f) Uba1 (ubiquitin-like modification activating enzyme 1) In the cerebrospinal fluid, Muse cells and MSCs suppressed expression on both postnatal days 5 and 14. The effect was faster with Muse cells than with MSCs (Figure 12B).

[0070] Example 6. Evaluation of the localization and expression changes of various protein molecules in the brain by immunofluorescence method (Methods) For the localization of various proteins, brains from 5-day-old rat pups (n = 5 / group) were used. Anesthetized rats were transcardially perfused with phosphate-buffered saline (PBS) and then fixed by perfusion with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brain samples were post-fixed with 4% PFA in 0.1 M phosphate buffer for 24 hours, then cryoprotected in phosphate-buffered sucrose containing 0.1% sodium azide (10% sucrose for 4-6 hours, 20% sucrose for 4-6 hours, and 30% sucrose for 12-36 hours) and flash-frozen at -80°C. After freezing, brain sections were cut coronally at a thickness of 40 μm. The sections were blocked with 10% normal goat serum for 1 hour at room temperature and then incubated with primary antibodies: rabbit anti-A2m (Abcam; ab58703, 1:50), rabbit anti-CD200 (Proteintech; 14057-1-AP, 1:50), rabbit anti-Ube1 (Uba1) (Proteintech; 15912-1-AP, 1:50), or rabbit anti-neuroserpin (Abcam; ab33077, 1:100) for 20 hours at 4°C. After three 5-minute washes with PBS, the sections were incubated with ATTO 488-conjugated secondary antibody: goat anti-rabbit IgG (Rockland; 611-152-122, 1:500) for 12 hours at 4°C. After washing three times with PBS for 5 minutes, the sections were incubated for 20 hours at 4°C with primary antibodies: mouse anti-NueN (Merck: MAB377, 1:100), mouse anti-Olig2 (Proteintech: 66513-1-Ig:14057-1-AP, 1:100), mouse anti-S100 (Abcam: ab4066, 1:100), or mouse anti-Iba1 (Abcam: ab15690, 1:100). These antibodies were used as markers for neurons, oligodendrocytes, astrocytes, and microglia, respectively. After washing three times with PBS for 5 minutes, the sections were incubated for 12 hours at 4°C with secondary antibodies: ATTO 550-conjugated goat anti-mouse IgG (Rockland, 611-152-122, 1:500). After washing with PBS three times for 5 minutes, the sections were passed once in purified water and then mounted while nuclear staining was performed using Prolong Gold with DAPI.After drying, the brain was observed using a confocal laser microscope (TiE-A1R, Nikon, Japan). Ten sections were selected from the nasal direction from the intersection of the sagittal and coronal sutures (Bregma) of the brain, and the hippocampus (Fig. 13A), striatum (Fig. 14A), and cerebral cortex (Fig. 15A) of each section were observed. One hundred cells were randomly selected from each brain region, and the expression levels of four candidate biomarker proteins in NeuN-, Olig2-, S100-, and Iba1-positive cells were quantified relative to their fluorescence intensity. The cells with the highest intensity of the four candidate proteins were assigned a value of 100, and non-expressing cells were assigned a value of 0.

[0071] (Results) As shown in Figures 13B-E, 14B-E, and 15B-E, neuroserpin was strongly expressed in NeuN-positive cells (neurons) in both the sham-operated and FGR groups, but was barely expressed in other cells. Furthermore, its expression intensity was significantly elevated in the FGR group. A2m was generally not strongly expressed in the sham-operated group, whereas its high expression was observed in NeuN-positive cells, S100-positive cells (astrocytes), and Olig2-positive cells (oligodendrocytes) in the FGR group. CD200 was strongly expressed in S100-positive cells in both the sham-operated and FGR groups, followed by NeuN-positive cells. In these cells, CD200 expression was more pronounced in the FGR group than in the sham-operated group. Ubb was detected in NeuN-positive cells, S100-positive cells, and Olig2-positive cells, and its expression intensity was significantly elevated in the FGR group. These results were similar in all brain regions: the cerebral cortex, hippocampus, and striatum. These results revealed that the expression of the four candidate biomarker proteins was significantly elevated in brain tissues. Furthermore, it was shown that the expression of the candidate proteins depended on the type of brain cells, not on the brain region. [Industrial Applicability]

[0072] The biomarkers of the present invention can be used for the early diagnosis of neonatal perinatal disorders resulting from fetal growth restriction in mammalian subjects and can be applied to the treatment of neurodevelopmental disorders in infants and children.

[0073] All publications and patents cited herein are incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, those skilled in the art will readily understand that various modifications may be made therein without departing from the spirit and scope of the invention.

Claims

1. 1. A method for aiding in the early diagnosis of neonatal perinatal disorders resulting from fetal growth restriction in a mammalian subject, comprising: (a) testing the expression level of one or more proteins selected from the group consisting of alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6) in a body fluid sample obtained from the subject, compared to expression levels in normal body fluids, body fluids after treatment, or body fluids known to be indicative of perinatal disorders; and (b) When the expression level shows a statistically significant difference compared to the expression level in the normal body fluid or the body fluid after treatment, or when the expression level does not show a statistically significant difference compared to the expression level in the body fluid known to indicate a perinatal disorder, the expression level is used as an indicator for diagnosing a perinatal disorder in the subject. wherein the body fluid is blood or cerebrospinal fluid.

2. 2. The method of claim 1, wherein the protein is selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini 1).

3. The method of claim 1 or 2, wherein the subject is a human patient.

4. The method according to any one of claims 1 to 3, wherein if all of the tested proteins show a significant difference in the body fluid sample of the subject compared to normal body fluid, this is used as an indicator for diagnosing a perinatal disorder in the subject.

5. The method of any one of claims 1 to 4, wherein the expression level is determined by immunoassay.

6. The method of any one of claims 1 to 4, wherein the expression level is determined by liquid chromatography / mass spectrometry.

7. The method of any one of claims 1 to 4, wherein the expression level is determined using a protein array.

8. Use of a proteomic profile of expression of one or more proteins selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), neuroserpin (Serpini1), ubiquitin thioesterase OTUB1 (Otub1), and ubiquitin-like modification activating enzyme 1 (Uba1) in blood or cerebrospinal fluid obtained from a mammalian subject for early assessment of perinatal disorders in newborns resulting from fetal growth restriction in the subject.

9. 9. The use according to claim 8, wherein the protein is selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpinil).

10. A method for assisting in the early determination of the effectiveness of a treatment for perinatal disorders in newborns caused by fetal growth restriction in a mammalian subject, comprising: (a) testing the expression level of one or more proteins selected from the group consisting of alpha-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neuroserpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6) in a body fluid sample obtained from a subject undergoing treatment, compared to expression levels in normal body fluids, body fluids after treatment, or body fluids known to be indicative of a perinatal disorder; and (b) When the expression level does not show a statistically significant difference compared to the expression level in the normal body fluid or the body fluid after treatment, or shows a statistically significant difference compared to the expression level in the body fluid known to indicate a perinatal disorder, the expression level is used as an index for determining that treatment of a perinatal disorder in the subject is effective. wherein the body fluid is blood or cerebrospinal fluid.

11. 11. The method of claim 10, wherein the protein is selected from the group consisting of alpha-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1).

Images