Biomarkers of pregnancy loss

Abstract

The present invention relates to methods for assessing the risk of pregnancy loss or embryo implantation failure, and also to methods for monitoring or assessing the effect of treatment using specific biomarkers to reduce the risk of pregnancy loss or embryo implantation failure. The invention also relates to the use of these biomarkers in a method of diagnosing a reproductive disorder in an individual, and also to a method of treating a reproductive disorder. In addition, the biomarkers may also be used in methods of selecting patients receiving treatment to reduce the risk of embryo implantation failure or abortion. The invention also relates to a kit for use in any of the methods described herein.

Description

Technical Field

[0001] The present invention relates to methods for assessing the risk of pregnancy loss or embryo implantation failure, and also to methods for using specific biomarkers to monitor or evaluate the effects of treatment to reduce the risk of pregnancy loss or embryo implantation failure. The present invention also relates to the use of these biomarkers in methods for diagnosing reproductive disorders in individuals, and also to methods for treating reproductive disorders. In addition, the biomarkers can also be used in methods for selecting patients for treatment to reduce the risk of embryo implantation failure or miscarriage. The present invention also relates to kits for use in any of the methods described herein. Background Art

[0002] Approximately 15% of clinical pregnancies end in pregnancy loss. 1 , most often occurring in early pregnancy. Fetal chromosomal abnormalities account for 50-60% of miscarriages 2,3 The incidence of aneuploid pregnancies is increasing in developed countries, which is in line with the demographic trend of aging women of childbearing age. 2 Aneuploidy is a rare condition in recurrent pregnancy loss (RPL). 4-8 , the latter being defined as two or more losses 9,10 With each additional pregnancy loss, the frequency of euploidy loss increases, and the likelihood of a successful pregnancy decreases. 4 Although these observations suggest that maternal factors contribute to higher-order pregnancy losses, few interventions have been shown to improve live birth rates in RPL. 9 , which reflects that in many cases the underlying mechanisms are still unclear.

[0003] Growing evidence from animal studies suggests that dysregulated interactions between the conceptus and the endometrium at the time of implantation can trigger a chain reaction that ultimately leads to pregnancy failure. 11-13 Implantation requires dramatic remodeling of the endometrial stroma, driven by the postovulatory progesterone surge and the rise in intracellular cyclic adenosine monophosphate levels. 14 This process, called decidualization, begins during the mid-luteal phase of each cycle and involves the differentiation and polarization of endometrial stromal cells (EnSCs) into stress-resistant and stress / senescent decidual subpopulations. 15 In parallel, uterine natural killer (uNK) cells accumulate in the stroma and, in response to IL-15 activation, target stressed decidual cells for clearance. After implantation, embryo-derived chorionic gonadotropin rescues the corpus luteum, while persistent progesterone signaling promotes the formation of tight junctions in the immune-privileged decidual stroma surrounding the conceptus. 16Therefore, the key challenge at implantation is to simultaneously avoid impending endometrial destruction while transforming the cycling endometrium into a semipermanent tissue (decidua) that is maintained throughout pregnancy.

[0004] The balance between decidual subpopulations is controlled by extrauterine cells, primarily natural killer (NK) cells and bone marrow-derived mesenchymal stem cells (MSCs). Figure 1 In short, the balance of decidual subpopulations from cycle to cycle at the time of implantation is regulated by the influx of MSCs, enabling expansion of the decidual cell pool and the uterine NK cell pool in early pregnancy, which targets and clears stressed / senescent cells. Recurrent pregnancy loss is associated with both MSC and uNK cell deficiencies 15,18-19 (Brighton et al., 2017, Lucas et al., 2016, Lucas et al., 2020). Importantly, the level of depletion is associated with the number of previous miscarriages and, in turn, the risk of recurrence. 18,20 (Lucas et al., 2016, Tewary et al., 2020).

[0005] Pre-pregnancy screening and intervention could reduce the burden of pregnancy loss. However, there is currently no diagnostic test to (i) assess the role of endometrial dysfunction in recurrent pregnancy loss, (ii) evaluate the efficacy or therapeutic intervention before pregnancy, and (iii) inform patients and their physicians of the risk of future loss in chromosomally normal pregnancies. The current implantation clinic at the Biomedical Research Unit in Reproductive Health (UHCW) offers a test based on the quantification of uterine NK cells in mid-luteal endometrial biopsies using immunohistochemistry (CD56 staining) and image analysis. Due to the inherent variability in endometrial uNK cell levels, biopsies obtained over two menstrual cycles are tested. Patients are self-referral or referred by their GP. The diagnostic pathway is complex, labor-intensive, and relies on experienced staff. Therefore, the results of the test are not available until 5-6 weeks later. Another important disadvantage of current uNK cell testing is that it relies on indirect measurements of the "state" of the endometrium.

[0006] However, a major challenge in the clinical management of recurrent pregnancy loss is distinguishing between embryonic and maternal causes of pregnancy loss. Two independent risk factors, maternal age and number of prior pregnancy losses, have a disproportionate impact on the rate of pregnancy loss (Magnus et al., 2019). The risk of pregnancy loss is age-related because the incidence of meiotic errors in oocytes increases, leading to a rapid increase in aneuploid embryos after age 35. On the other hand, the risk of recurrent pregnancy loss increases progressively by approximately 10% with each additional pregnancy loss, independent of maternal age (Magnus et al., 2019). Therefore, a fundamental principle for any clinical test designed to identify maternal factors contributing to pregnancy loss is that the frequency of positive test results must increase with each additional pregnancy loss, regardless of maternal age. Currently, no pregnancy loss test in clinical practice has been shown to meet this basic criteria for causality.

[0007] The lack of decidual cells and the excess of stressed / senescent stromal cells provide an endometrial environment that is easily invaded by the embryo, lacks biosensing properties, and is easily damaged. At the functional level, this pathological endometrial state is called "implantation checkpoint failure", which means that the endometrium can neither select low-fitness embryos nor adequately support high-quality embryos. Both situations lead to clinical pregnancy loss. 15,22-24 (Brosens et al., 2014, Ewington et al., 2019, Brighton et al., 2017, Brosens et al., 2022).

[0008] Recent breakthroughs in single-cell RNA sequencing (scRNA-seq) have led to the discovery of novel biomarkers for specific endometrial stromal and epithelial subpopulations during the peri-implantation window. The inventors have previously found (as described in WO2021 / 032973) that abnormal levels of markers for decidual cells and / or senescent decidual cells are associated with reproductive defects (e.g., miscarriage, particularly SCARA5 and DIO2). Summary of the Invention

[0009] The discovery of specific biomarkers for decidua and decidual senescent cells enables the assessment of the "state" of the endometrium during the luteal phase implantation window prior to pregnancy and the identification of women at risk for subsequent pregnancy loss due to impaired endometrial function. The inventors unexpectedly discovered that PLA2G2A is a biomarker for pregnancy loss, particularly recurrent pregnancy loss. The biomarkers of the present invention are also advantageous when compared to previously described biomarkers (e.g., SCARA5) due to the greater dynamic range in PLA2G2A expression (e.g., Figure 2B) with enhanced sensitivity. The development of clinical tests based on the biomarkers described herein, including PLA2G2A testing, can be used for the diagnosis and management of women with a history of one or more prior pregnancy losses. The tests can also be used to evaluate the efficacy of pre-pregnancy interventions, including drug therapies, aimed at reducing the risk of pregnancy loss and increasing live birth rates.

[0010] The test of the present invention is specifically designed to assess the endometrial causes of pregnancy loss before pregnancy and to assist / guide therapeutic intervention. Due to its simplicity and easiness, it has the potential for becoming a routine, rapid and relatively inexpensive test for any woman suffering from pregnancy loss (even for the first time). Compared with the currently available commercial tests (described in EP2333107B1) intended to determine the "implantation window" in IVF patients, it is a simpler and cheaper alternative. Importantly, the test of the present invention shows that the frequency of positive results increases with each additional pregnancy loss, regardless of maternal age, so it can be said that the maternal factors associated with miscarriage can be identified.

[0011] Therefore, the inventors unexpectedly discovered that PLA2G2A can be used alone or in combination as a biomarker for assessing the risk of pregnancy loss or embryo implantation failure, and can also be used to monitor or evaluate the impact of treatment to reduce the risk of pregnancy loss or embryo implantation failure. The biomarkers described herein can also be used in methods for diagnosing reproductive disorders in individuals and can also be used in methods for assisting the treatment of reproductive disorders. In addition, these biomarkers can also be used in methods for selecting patients for treatment to reduce the risk of embryo implantation failure or miscarriage, and methods for stratifying patients. The biomarkers can be detected using the kits described herein.

[0012] Therefore, the present invention provides a method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene of decidual senescent cells and at least one marker gene of decidual cells in a biological sample obtained from the individual, thereby assessing the risk, wherein the at least one marker gene of decidual cells comprises PLA2G2A.

[0013] The present invention also provides a method for monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effect of the treatment, wherein the at least one marker gene of the decidual cells comprises PLA2G2A.

[0014] The present invention also provides a method for diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells in a biological sample obtained from the individual, thereby diagnosing the disorder, wherein the at least one marker gene of the decidual cells comprises PLA2G2A.

[0015] The present invention also provides methods of treating a reproductive disorder in an individual or preventing pregnancy loss or embryo implantation failure in an individual, the methods comprising diagnosing a reproductive disorder or assessing the risk of pregnancy loss or embryo implantation failure as described herein, and administering an agent or implementing a treatment regimen to effectively treat the reproductive disorder or prevent pregnancy loss or embryo implantation failure in an individual diagnosed positive or assessed as being at risk.

[0016] The present invention also provides a method for selecting a patient for treatment to reduce the risk of embryo implantation failure or miscarriage, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells in a biological sample obtained from an individual, and selecting the patient for treatment to reduce the risk of pregnancy loss or embryo implantation failure based on the levels of the marker genes, wherein the at least one marker gene of decidual cells comprises PLA2G2A.

[0017] The present invention also provides a detection kit suitable for the method described herein, wherein the detection kit includes a device for detecting or quantifying at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells at the nucleic acid or protein level, and optionally a device for detecting and / or quantifying the level of uNK cells in the individual or the level of at least one marker gene of uNK cells.

[0018] The present invention also provides a method for assessing readiness for pregnancy or successful embryo implantation in an individual, comprising detecting and / or quantifying the amount of at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells in a biological sample obtained from the individual, thereby assessing readiness for pregnancy or successful embryo implantation, wherein the at least one marker gene of the decidual cells comprises PLA2G2A.

[0019] The present invention provides a highly advantageous endometrial test for patients with recurrent pregnancy loss. The test is suitable for any woman planning a pregnancy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Mechanisms affecting the decidual pathway

[0021] The transformation of the cyclical endometrium into the pregnant decidua requires the coordinated clearance of senescent decidual cells by decidual cells and uterine natural killer (uNK) cells. Simultaneously, bone marrow-derived mesenchymal stem cells (MSCs) are recruited to ensure rapid tissue expansion during pregnancy. A deficiency in MSCs and / or uNK cells drives a pro-senescent decidual response, rendering the uteroplacental interface susceptible to tissue disruption during pregnancy and leading to miscarriage. SASP, senescence-associated secretory phenotype; IL-15, interleukin-15.

[0022] Figure 2 : Expression of decidual cells and decidual senescent cell markers in the endometrium

[0023] (A) SCARA5 and DIO2 percentiles for 6 paired endometrial biopsies (n=12) by RNA-seq. Open and solid circles represent "A" and "B" biopsies, respectively. Paired biopsies are indicated by dotted lines. Samples in the upper left quadrant (SCARA5 mRNA levels <30th percentile and DIO2 >70th percentile) were considered "abnormal," i.e., harboring an excess of stress / senescent cells and lacking decidual cells. (B) Volcano plot indicating gene expression differences between samples considered "normal" and "abnormal" endometrial samples showed that high DIO2 expression was associated with low PLA2G2A expression, and vice versa. The Bonferroni correction at 0.05 is indicated by the dotted line. (C) Violin plot showing DIO2 expression and PLA2G2A mRNA expression in endothelial, epithelial, immune, and stromal cells from single cells in vivo in luteal phase endometrial biopsies. (D) DIO2 and PLA2G2A mRNA expression in the proliferative phase and early, mid, and late luteal endometrium. Each column represents a separate biopsy. The data were retrieved from a microarray dataset stored in the Gene Expression Omnibus (GEO Profile ID: GDS2052). (E) DIO2 and PLA2G2A mRNA levels were quantified by RT-qPCR analysis in 822 endometrial biopsies obtained between LH+6 and LH+11. Percentile plots showing the distribution of gene expression throughout the peri-implantation window were generated based on normalized expression values using R software. The median number of samples per day was 153 (range: 51-202).

[0024] Figure 3 : Spatial organization of cells expressing PLA2G2A and DIO2 in the endometrium

[0025] Dot plots depict spatially log-transformed normalized expression of DIO2 (upper panel) and PLA2G2A (lower panel) in formalin-fixed, paraffin-embedded endometrial tissue sections (LH+8). Original magnification 10×.

[0026] Figure 4: Association of the PLA2G2A / DIO2 ratio with the number of previous miscarriages

[0027] (A) Distribution of PLA2G2A / DIO2 percentiles in 854 LH phase endometrial biopsies from patients with 0 to 18 prior miscarriages. Different letters above the columns indicate significance at P < 0.05 after one-way ANOVA with Tukey's multiple comparison test. (B) Frequency of endometrial biopsies with a PLA2G2A / DIO2 ratio in the lower quartile as a function of number of prior miscarriages. (C) Frequency of endometrial samples with a PLA2G2A / DIO2 ratio < 15th percentile as a function of number of prior miscarriages. The total number n for each column is indicated. Different letters above the columns indicate significance at P < 0.05 after a chi-square test with repeated measures. (D) Comparison of age (upper panel), BMI (middle panel), and biopsy day after the luteinizing hormone surge (LH+, lower panel) between samples with a PLA2G2A / DIO2 ratio >15th percentile (designated "normal") and <15th percentile ("abnormal"). Student's t-test; ** indicates P < 0.01; ns: not significant (P > 0.05).

[0028] Figure 5 Using the PLA2G2A / DIO2 ratio to predict risk of future pregnancy loss

[0029] (A) Analysis of the PLA2G2A / DIO2 ratio in endometrial biopsies obtained before pregnancies that resulted in live birth or miscarriage. Lower than The relative proportion of live births to pregnancy losses in pre-pregnancy endometrial biopsies with the indicated percentiles of the PLA2G2A / DIO2 ratio. Higher than Relative proportions of live births versus pregnancy losses in pre-pregnancy endometrial biopsies with the indicated percentiles of the PLA2G2A / DIO2 ratio. Chi-square test, * indicates P < 0.05. ns: not significant (P > 0.05). (B) Age, body mass index (BMI), and endometrial uNK cell percentiles before subsequent pregnancies that resulted in live birth or miscarriage. Student's t-test; ns: not significant (P > 0.05).

[0030] Figure 6 : Effects of oral sitagliptin on the endometrial PLA2G2A / DIO2 ratio

[0031] In the placebo (n=16) and sitagliptin (n=15) groups of the SIMPLANT trial (EudraCT No. 2016-001120-54), DIO2 and PLA2G2A transcripts were detected by RT-qPCR in paired baseline and second endometrial biopsies obtained from participants. The PLA2G2A / DIO2 ratio was calculated (bottom panel). Data were analyzed using the Wilcoxon paired signed rank test with Sidak correction. DETAILED DESCRIPTION

[0032] It should be understood that different applications of the disclosed method can be customized for specific needs in the art. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments of the present invention and are not intended to be restrictive. All publications, patents and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes "cells," etc.

[0034] Methods used to assess the risk of pregnancy loss or embryo implantation

[0035] The present invention provides a method for assessing the risk (or likelihood or probability) of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, thereby assessing the risk, wherein the at least one marker for the decidual cells comprises PLA2G2A. In the above method and all other methods and aspects of the present invention, PLA2G2A is detected as the decidual marker gene. In other words, the at least one decidual marker gene detected is PLA2G2A. Thus, the decidual marker genes used for detection include at least PLA2G2A. The method may also comprise detecting additional decidual marker genes.

[0036] Any additional marker genes for decidual cells and the at least one marker gene for decidual senescent cells can be selected from any such marker genes. Thus, the marker gene can be any gene that indicates the level of decidual cells and decidual senescent cells in a sample (e.g., an endometrial sample). For example, the marker gene for decidual cells can be any marker whose decrease indicates a decrease in the level of decidual cells, and the marker gene for decidual senescent cells can be any marker whose increase indicates an increase in the level of decidual senescent cells. For example, the additional marker gene for decidual cells can be selected from SCARA5, FTL, GLRX, and / or IL1RL1. The at least one marker gene for decidual senescent cells can preferably include DIO2. The at least one marker gene for decidual senescent cells can be selected from DIO2, CLU, and IGFBP1.

[0037] The level of marker gene is usually compared with control sample or reference sample or level. Any suitable control sample or reference sample or level can be used. Control sample or reference sample or level can represent normal or healthy sample / level, for example, from the individuality that does not suffer from any reproductive disorder or does not suffer from pregnancy loss or embryo implantation failure or multiple individuals obtain or determine. Individuality can have one or more successful pregnancies. Control or reference sample or level can also be obtained or determined from the individuality that has actively responded to treatment to reduce the risk of pregnancy loss or embryo implantation failure or multiple individuals. Alternatively, control sample or reference sample or level can represent the sample / level from the individuality that suffers from reproductive disorder or has one or more miscarriages or embryo implantation failure or multiple individuals, i.e. positive control or reference sample or level. In the case of the control or reference sample or level from multiple individuals, mean value can be obtained or the results can be gathered to produce a more accurate reference range. The level determined in the test sample is preferably compared with the control or reference sample or level obtained as the test sample on the same day or in recent days of the menstrual cycle. Those skilled in the art can easily identify other suitable controls or reference samples or levels.

[0038] The level of a marker gene (e.g., PLA2G2A) for decidual cells that is decreasing or has decreased in a sample compared to a reference sample or level can indicate that the individual is at risk for pregnancy loss or embryo implantation failure. Alternatively, the level of a marker gene (e.g., DIO2) for decidual senescent cells that is increasing or has increased compared to a reference sample or level can indicate that the individual is at risk for pregnancy loss or embryo implantation failure. The level of the marker gene can be determined using any method described herein and known to those skilled in the art. In one example, the level of a marker gene (e.g., PLA2G2A) for decidual cells that is decreasing or has decreased compared to a reference sample or level, and the level of a marker gene (e.g., DIO2) for decidual senescent cells that is increasing or has increased compared to a reference sample or level, together indicate that the individual is at risk for pregnancy loss or embryo implantation failure.

[0039] In one example, the method for assessing risk of pregnancy loss or embryo implantation failure in an individual further comprises detecting and / or quantifying the level of uterine natural killer (uNK) cells in the sample, for example based on the level of at least one marker gene for uNK cells in the sample.

[0040] Decreasing or decreased levels of uNK cells or uNK cell gene markers in a sample compared to a reference sample or level may indicate that the individual is at risk for pregnancy loss or embryo implantation failure.

[0041] The methods of the present invention may further comprise detecting and / or quantifying genes that allow identification of days in the menstrual cycle, as described below.

[0042] Further risk markers that can be used to assess the risk of pregnancy loss or embryo implantation failure include maternal body mass index (BMI), maternal age, number of previous pregnancy losses or embryo implantation failures, familial and generational factors, infertility history, placental abnormalities, cervical and uterine abnormalities, smoking, alcohol consumption, etc. Additional risk markers are known to those skilled in the art.

[0043] For example, women may be at increased risk for pregnancy loss or implantation failure if their maternal BMI is too low (e.g., <18.5) or too high (e.g., >25), if their maternal age is 35 or older, or if they have a history of recurrent pregnancy loss (e.g., when a woman has had two or more pregnancy losses before reaching 20 weeks of gestation).

[0044] sample

[0045] As used herein, the term "biological sample" or "sample" refers to any sample taken from an individual. In the context of the methods of the present invention, suitable samples include, for example, endometrial tissue, endometrial secretions, cells obtained from the endometrium, or an endometrial biopsy.

[0046] The sample obtained from the endometrium can be collected by any method known in the art, including by endometrial biopsy or endometrial sampling. This technique involves removing a piece of tissue from the inner lining of the uterus (endometrium). A sample can also be obtained using a dilation and curettage procedure.

[0047] The sample may be or has been processed before use, for example by dilution, centrifugation or extraction of DNA, RNA or protein. The sample may be a freshly obtained sample, or may be or have been stored or preserved, for example by freezing, before use.

[0048] Samples can be collected during the luteal phase of the menstrual cycle. The luteal phase begins with the formation of the corpus luteum, and progesterone is significantly higher than other stages of the menstrual cycle. Samples can be collected during the mid-luteal phase of the menstrual cycle. Therefore, samples are usually collected after ovulation. Samples are usually collected during the embryo implantation window (also referred to as the receptivity window) of the menstrual cycle, during which the endometrium can accommodate embryo implantation. The embryo implantation window can be determined by any means and can be calculated, for example, based on an ovulation test. The ovulation test can be based on hormone levels, such as luteinizing hormone (LH) levels (such as LH levels in urine) or estrogen levels (such as based on saliva crystallization). Alternatively, the level of one or more markers indicating acceptance of embryo implantation can be determined. Samples can be collected between about 5 days and about 11 days after LH levels increase or surge (such as 2-5 times increase or surge) (i.e. LH+5 to LH+11). LH is produced by the pituitary gland and is usually secreted at very low levels throughout the menstrual cycle, but the ovulation phase of the menstrual cycle begins with a surge in LH.

[0049] The above-mentioned sample types and sampling timings are applicable to any method of detecting marker levels of the present invention.

[0050] individual

[0051] The subject referred to in any of the methods of the present invention may be a human or non-human menstruating mammal. Thus, the methods described herein may be applied in a veterinary setting. The subject is preferably a human female.

[0052] An individual may have or have had infertility or embryo implantation failure. For example, an individual may have or have had embryo implantation failure following in vitro fertilization treatment. An individual may have at least one prior pregnancy loss or multiple pregnancy losses and / or at least one prior embryo implantation failure or multiple embryo implantation failures. An individual may have recurrent pregnancy loss (RPL).

[0053] Individuals may already be considered to be at risk for pregnancy loss or embryo implantation failure. Due to the presence of one or more risk markers, including low or high body mass index (BMI), maternal age, number of previous pregnancy losses or embryo implantation failures, familial and generational factors, infertility history, placental abnormalities, cervical and uterine abnormalities, etc., individuals may be considered to be at risk for pregnancy loss or embryo implantation failure. Additional risk markers for pregnancy loss or embryo implantation failure are known to those skilled in the art.

[0054] Pregnancy loss

[0055] Pregnancy loss refers to the inability of an embryo to produce a baby, which may be caused by unsuccessful embryo implantation. Pregnancy loss assessed according to the biomarkers of the present invention is typically a loss in early pregnancy, particularly in the first 20 to 23 weeks of pregnancy, which may also be referred to as "miscarriage". Typical symptoms of miscarriage include vaginal bleeding with or without pain, and cramping and pain in the lower abdomen. When at least two or more miscarriages occur, an individual may be diagnosed with recurrent pregnancy loss (RPL) or infertility. However, RPL may not be a binary condition, and there may be an increased risk of further pregnancy loss with any previous loss. The present invention allows for the detection of any susceptibility to RPL, regardless of the number of previous losses.

[0056] Most miscarriages are thought to be caused by chromosomal abnormalities or errors in the embryo, such as aneuploidy, for example, autosomal trisomy, monosomy X, triploidy, tetraploidy, etc. Other miscarriages are not due to chromosomal abnormalities or errors in the embryo.

[0057] In the methods of the present invention, the risk of pregnancy loss / miscarriage can be assessed. The methods of the present invention are preferably used to determine the risk of euploid miscarriage, and wherein the pregnancy loss is not due to a chromosomal abnormality or error in the embryo. The methods of the present invention are preferably used to determine the risk of recurrent pregnancy loss / miscarriage.

[0058] Embryo implantation failure

[0059] After fertilization, the fertilized egg (or zygote) begins to produce an embryo through mitosis. The process by which the embryo attaches to the inner layer of the uterus (i.e., the endometrium) is called implantation. "Embryo implantation failure" or "implantation failure" according to the present invention refers to the failure of the embryo to implant into the endometrium. Implantation failure may occur when a patient attempts to become pregnant naturally without receiving any fertility treatment, or after undergoing assisted reproductive technology such as in vitro fertilization (IVF).

[0060] Some cases of implantation failure are thought to be caused by chromosomal abnormalities in the embryo, such as aneuploidy, e.g., autosomal trisomy, monosomy X, triploidy, tetraploidy, etc. Other cases of embryo implantation failure are not due to chromosomal abnormalities or errors in the embryo. Some cases of implantation failure associated with IVF are caused by poor embryo quality, the age of the egg, lack of response to IVF medications, or other lifestyle factors (e.g., smoking).

[0061] According to the present invention, the risk of any embryo implantation failure case can be assessed in any application scenario. Thus, the method of assessing the risk of embryo implantation failure of the present invention can be used to determine the risk of implantation failure following natural conception or, in an individual, the risk of implantation failure following assisted reproduction, such as in vitro fertilization. The method is preferably used to determine the risk of embryo implantation failure that is not due to chromosomal abnormalities or errors in the embryo.

[0062] Decidual cells, decidual senescent cells and marker genes

[0063] According to the method for assessing the risk of pregnancy loss or implantation failure of the present invention, at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells are detected and / or quantified, wherein the at least one marker gene of decidual cells comprises PLA2G2A.

[0064] When exiting the cell cycle, cells differentiate into specialized cells or become senescent. Cellular senescence is a defined cellular state that can be acute or chronic. The key characteristic of senescent cells (SNC) is that they are in a state of permanent cell cycle arrest, typically initiated and maintained by the p53-p21-retinoblastoma (RB) and p16-RB tumor suppressor pathways. SNC produce a bioactive "secretome" called the senescence-associated secretory phenotype (SASP), which can disrupt normal tissue architecture and function through multiple mechanisms, including recruiting inflammatory immune cells, remodeling the extracellular matrix, inducing fibrosis, and inhibiting stem cell function. 58 .

[0065] During decidualization, cells of the endometrium undergo significant changes in preparation for and during pregnancy. During this process, endometrial stromal cells (EnSCs) become specialized cells (i.e., decidual cells) or become acutely senescent (i.e., decidual SNCs).

[0066] The decidual cells detected can be any decidual cells, and the decidual senescent cells detected can be any decidual senescent cells, typically any such cells in an endometrial sample. The decidual or decidual senescent cells are typically derived from endometrial stromal cells. Decidual cells are stress-resistant and are also described herein as stress-resistant decidual cells.

[0067] According to the method for assessing the risk of pregnancy loss or implantation failure of the present invention, at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells can be detected, wherein the at least one marker gene of decidual cells comprises PLA2G2A. The at least one marker gene of decidual cells may further comprise one or more of SCARA5, FTL, GLRX, and IL1RL1, and the marker genes of decidual senescent cells typically comprise one or more of DIO2, CLU, and IGFBP1. The method may include detecting a decrease in PLA2G2A. Preferably, the at least one marker gene of decidual senescent cells is DIO2. The method may include detecting an increase in DIO2. In a preferred embodiment, the at least one marker gene of decidual senescent cells is DIO2, and the method includes detecting and / or quantifying the amounts of both PLA2G2A and DIO2. The method may include detecting a decrease in PLA2G2A and an increase in DIO2. Any of the above genes can be detected and / or quantified in the method for assessing the risk of pregnancy loss or embryo implantation failure of the present invention, as well as in further methods described below.

[0068] Percentile graphs (centile or percentile graphs) can be used to compare the expression levels of marker genes in samples obtained on different days in the menstrual cycle, such as the marker genes of decidual and decidual senescent cells (and the levels of other labels as described below). Percentile graphs are based on the statistical distribution of the expression levels of given marker genes on a given day in the menstrual cycle (e.g., after a positive ovulation test). The more samples used to generate percentile graphs, the more accurate the reference range. For example, percentile graphs can be based on at least 10 samples, at least 100 samples, at least 250 samples, at least 500 samples, at least 1000 samples, at least 2000 samples, at least 5000 samples or more. The relative expression levels of given marker genes (i.e., percentiles) in individual samples obtained on a given day in the menstrual cycle can be calculated with reference to percentile graphs.

[0069] In some cases, determining the percentile of each marker gene allows determination of individual causes and clinical manifestations, such as for recurrent pregnancy loss. A putative defect or decidual dyshomeostasis of the decidual pathway can be determined. In one embodiment, low levels of decidual cell marker genes (e.g., PLA2G2A) and high levels of decidual senescent cell marker genes (e.g., DIO2) indicate excessive decidual aging compared to a reference sample or reference level, and occur more frequently in recurrent pregnancy loss. In certain cases, if the ratio of PLA2G2A to DIO2 is lower than the 50th percentile compared to a reference sample or reference level, a positive diagnosis is present. In other cases, if the ratio of PLA2G2A to DIO2 is lower than the 40th percentile, the 30th percentile, the 20th percentile, or the 10th percentile compared to a reference sample or reference level, a positive diagnosis is present.

[0070] In other cases, the present invention can include determining the relative risk of miscarriage. For example, it can be determined that an individual is twice as likely to have a miscarriage as to have a live birth. In other aspects, it can be determined that an individual is three, four, or five times more likely to have a miscarriage. In one particular case, when the ratio of PLA2G2A to DIO2 is below the 15th percentile, it can be determined that an individual is twice as likely to have a miscarriage.

[0071] uNK cells and uNK cell gene markers

[0072] According to the method for assessing the risk of pregnancy loss or implantation failure of the present invention, in addition to the marker genes of decidual cells and decidual senescent cells, the level of uterine natural killer (uNK) cells can generally be detected and / or quantified by detecting one or more marker genes of uNK cells.

[0073] The successful transformation of the endometrium (from a cyclical tissue to a semi-permanent tissue that can maintain the placenta throughout pregnancy) depends on stress-resistant decidual cells, which mobilize (co-opting) uterine natural killer (uNK) cells to clear their acute stress counterparts, i.e., decidual senescent cells, by granule exocytosis. Therefore, the balance of decidual cell populations and uNK cells that differentiate during the mid-luteal phase of the menstrual cycle described herein may determine the ability of the endometrium to transform into pregnancy tissue. Imbalances in decidual subpopulations are also associated with reproductive failure in this article. Therefore, determining the level of uNK cells in combination with detecting the levels of markers for decidual and decidual senescent cells provides additional information for assessing the risk of pregnancy loss or implantation failure and more broadly related to the diagnosis of reproductive disorders.

[0074] The level of uNK cells in a sample can be detected and / or quantified by any means known in the art. Immunohistochemistry and image analysis can be used to detect and / or quantify uNK cells. Alternatively, uNK cells can be detected based on the level of at least one marker gene of uNK cells in a sample.

[0075] According to the methods of the present invention, any uNK cell gene marker can be detected and / or quantified. The marker genes for uNK cells can be selected from NCAM1, KLRB1, KLRC1, GZMA, GZMB, IL2RB, and IL2RG. The above genes can be detected and / or quantified in the methods described herein for assessing the risk of pregnancy loss or embryo implantation failure, and also in further methods of detecting marker genes of the present invention as described below. In recurrent pregnancy loss, NK cell deficiency occurs more frequently.

[0076] Determining the timing of the menstrual cycle

[0077] According to the method of the present invention, the time (for example, point, stage or date) in the menstrual cycle to obtained sample can be determined in addition.Any parameter can be used, comprise any known hormone, mark or other parameters (comprising above-mentioned any hormone or mark), so that point, stage or date in the cycle are timed.Preferably, in the embryo implantation window, sample is obtained, and point, stage or date in the embryo implantation window are determined.Point, stage or date in the menstrual cycle are determined advantageously to allow sample to be compared with reference sample or the level representing same point, stage or date, because hormone level or marker gene level change in whole cycle.

[0078] In a specific embodiment, in addition to the marker genes of decidual cells, decidual senescent cells and / or uNK cells, marker genes that allow identification of points, stages or dates in the menstrual cycle are also detected and / or quantified. Such marker genes are also referred to herein as molecular timing genes and generally indicate timing in the implantation window. By analyzing these genes, the accuracy of detection based on analysis of marker genes for decidual cells and decidual senescent cells and / or uNK cells or uNK cell marker genes can be improved.

[0079] The purpose of molecular timing is twofold. Due to the cycle-dependence of gene marker levels, the knowledge of the biopsy sampling date in the cycle is advantageously used to help interpret the levels of decidual cell gene markers (e.g., PLA2G2A), decidual senescent cell gene markers (e.g., DIO2), and / or uNK cell levels or the levels of uNK cell gene markers. In practice, this can also be achieved by arranging the biopsy time during the pre-ovulatory luteinizing hormone (LH) surge as described above. Therefore, the method of the present invention can be implemented in an individual by obtaining a sample at an appropriate time point after the LH surge as described above. However, by considering molecular timing based on the analysis of marker gene expression, the risk of biopsy timing deviation due to patient error and inherent variations between the exact time of LH surge and ovulation can be reduced.

[0080] The implantation window (also referred to as the receptivity window) is associated with the dramatic changes in gene expression in the glandular epithelium. Therefore, the method of the present invention can preferably be included in any marker gene detected with expression changes (and usually selectively expressed) in the glandular epithelium during the implantation window, and therefore can be reported at a point, stage or date in the embryo implantation window. This determination can preferably be based on two or more genes that selectively express in glandular body and show opposite expression profiles along with the carrying out of the menstrual cycle. The ratio of two or more such genes can be determined.

[0081] Marker genes that can determine the molecular timing of the embryo implantation window and that can be used according to the present invention include any one or more of GPX3, DPP4 (GPX3-like gene), SLC15A2, and CTNNA2 (SLC15A2-like gene). Preferably, the genes that allow the timing of the days in the menstrual cycle to be identified may comprise, consist of, or consist essentially of GPX3 and SLC15A2. Thus, the ratio of GPX3 to SLC15A2 can be determined. Because the molecular timing used in the present invention is generally based on genes that are selectively expressed in epithelial cells (e.g., GPX3 and SC15A2), while decidual cell and decidual senescent cell markers (e.g., PLA2G2A and DIO2) are selective stromal cell markers, molecular timing can also be used to diagnose asynchrony between hormonal responses in the epithelial and stromal regions. As described herein, as the luteal phase unfolds, GPX3 and SLC15A2 are regulated in opposite ways (i.e., GPX3 is rapidly upregulated, while SLC15A2 is rapidly downregulated). Thus, during the implantation window, the ratio of these two genes varies greatly from day to day. The ratio between GPX3 and SLC15A2 rises significantly between LH+5 and LH+11 days of the cycle, so the ratio between GPX3 and SLC15A2 can be matched to a specific day in the cycle. A specific GPX3 / SLC15A2 ratio can be matched to a specific day in the cycle based on a centile / percentile graph that is plotted based on the values obtained from a pooled reference sample for that specific day in the cycle, such as by detecting the LH surge (e.g., using a home ovulation kit). Preferably, if the GPX3 / SLC15A2 ratio in the test sample falls within the 25th to 75th percentiles of a reference percentile chart plotted against values obtained from pooled reference samples on a particular day in the cycle, and matches (i.e., coincides with) the date of the luteal phase as determined by detection of an LH surge (e.g., using a home ovulation kit), the accuracy of the biopsy timing can be considered improved, and the test result can be reported based on the day in the cycle as determined by detection of an LH surge (e.g., using a home ovulation kit). If the GPX3 / SLC15A2 ratio in the test sample falls outside the 25th to 75th percentiles of a reference percentile chart plotted against values obtained from pooled reference samples on a particular day in the cycle (i.e., discordant), or if the GPX3 / SLC15A2 ratio does not match the date of the luteal phase as determined by detection of the LH surge (e.g., using a home ovulation kit), the timing of the biopsy can be considered less than accurate, and the test result can be reported based on either the date in the cycle as determined by detection of the LH surge (e.g., using a home ovulation kit) or the molecular timing result.

[0082] Marker gene sequence

[0083] Disclosed herein are specific sequences of marker genes useful according to the present invention and their database accession numbers / identification numbers in the NCBI gene database, the Ensembl database, and the OMIM database. The gene sequences disclosed herein include reference to gene sequences available in these online sequence databases on June 16, 2019. Therefore, below is a list of marker genes with representative accession numbers (in brackets: NCBI gene database, followed by Ensembl database, followed by OMIM database) and alternative gene names (italics):

[0084] Phospholipase A2 group IIA: PLA2G2A (5320, ENSG00000188257, 172411) MOM1, PLA2, PLA2B, PLA2L, PLA2S, PLAS1, sPLA2

[0085] Scavenger receptor class A member 5: SCARA5 (286133, ENSG00000168079, 611306) Tesr, NET33, FLJ23907, MGC45780;

[0086] Ferritin light chain: FTL (2512, ENSG00000087086, 134790) LFTD, NBIA3, MGC71996;

[0087] Glutaredoxin: GLRX (2745, ENSG00000173221, 600443) GRX, GRX1;

[0088] Interleukin-1 receptor-like 1: IL1RL1 (9173, ENSG00000115602, 601203) T1, ST2, DER4, ST2L, ST2V, FIT-1, IL33R;

[0089] Iodothyronine deiodinase 2: DIO2 (1734, ENSG00000211448, 601413) D2, 5DII, SelY, DIOII, TXDI2;

[0090] Clusterin: CLU (1191, ENSG00000120885, 185430) CLI, AAG4, APOJ, CLU1, CLU2, KUB1, SGP2, APO-J, SGP-2, SP-40, TRPM2, TRPM-2, NA1 / NA2;

[0091] Insulin-like growth factor binding protein 1: IGFBP1 (3484, ENSG00000146678, 146730) AFBP, IBP1, PP12, IGF-BP25, hIGFBP-1;

[0092] Glutathione peroxidase 3: GPX3 (2878, ENSG00000211445, 138321) GPx-P, GSHPx-3, GSHPx-P;

[0093] Solute carrier family 15 member 2: SLC15A2 (6565, ENSG00000163406, 602339) PEPT2;

[0094] Dipeptidyl peptidase IV: DPP4 (1803, ENSG00000197635, 102720) CD26, ADABP, ADCP2, DPPIV, TP103;

[0095] Cateninα2: CTNNA2 (1496, ENSG00000066032, 114025) CAPR, CTNR, CAP-R, CT114, CDCBM9;

[0096] Interleukin-2 receptor subunit β: IL2RB (3560, ENSG00000100385, 146710) CD122, IL15RB, P70-75;

[0097] Interleukin-2 receptor subunit γ: IL2RG (3561, ENSG00000147168, 308380) P64, CIDX, IMD4, CD132, SCIDX, IL-2RG, SCIDX1;

[0098] Neural cell adhesion molecule 1: NCAM1 (4684, ENSG00000149294, 116930) CD56, NCAM, MSK39.

[0099] Detection and / or quantification of the amount / level of a biomarker

[0100] As used herein, the term "marker gene" or "biomarker" refers to a gene or gene fragment whose amount and / or detection change can be correlated with a specific physical condition or state. Specific marker genes used in the present invention are associated with the risk of pregnancy loss or embryo implantation failure and are also used in the methods described herein. Detection and / or quantification of such marker genes can be achieved by any means and is not limited to detection / quantification of nucleic acids. Marker genes can also be detected by their respective expression products, including expressed peptides, polypeptides, and proteins and fragments thereof.

[0101] As used herein, the terms "amount" or "level" used herein refer to the amount of a marker gene or its expression product that is detectable or measurable in a biological sample and / or a control or reference sample. The amount of a marker gene can be, for example, the amount of a nucleic acid or protein. The term may alternatively include combinations thereof. The amount or level of a marker gene can refer to the absolute amount or level of a biomarker. Alternatively, the relative level or amount of a marker can be assessed by comparing the level or amount of a marker gene in a sample from a subject with a control value or reference value. Alternatively, in some cases, the relative amount or level of a marker gene can refer to the concentration of the marker gene relative to the total amount or total level of the marker gene in the sample.

[0102] The level of marker genes can be detected and / or quantified by detecting nucleic acids (e.g., RNA). For example, the level of mRNA can be measured by reverse transcription quantitative polymerase chain reaction (RT-PCR, followed by qPCR). RT-PCR is used to produce cDNA from mRNA. When the DNA amplification process is carried out, cDNA can be used for qPCR determination to produce fluorescence. Compared with the standard curve, qPCR can produce absolute measurements, such as the number of mRNA copies per cell. Northern blots, microarrays, Invader determinations, and RT-PCR combined with capillary electrophoresis can be used to measure the expression level of mRNA in a sample.

[0103] In some embodiments, nucleic acid amplification methods can be used to detect polynucleotide biomarkers. For example, oligonucleotide primers and probes can be used for amplification and detection methods using nucleic acid substrates separated by any of a variety of well-known and established methodologies. Methods for amplifying nucleic acids include, but are not limited to, polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), thermophilic SDA (tSDA), Taqman-PCR, multiplex Taqman-PCR, Nanostring, targeted sequencing, digital PCR, or any suitable method known in the art. In a preferred embodiment, the level of marker genes can be detected and / or quantified by droplet digital qPCR (ddPCR).

[0104] The detection and quantification of marker genes in the methods of the present invention may also involve the use of an agent that specifically detects the expression product of the marker gene, such as a protein or peptide of interest. The agent may be an antibody or its functional equivalent (i.e., an anti-peptide antibody) that binds to the protein or peptide being analyzed. These antibodies can be used to perform immunoassays, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunoprecipitation, immunohistochemistry, immunofluorescence, protein dot blot, Western blot, turbidimetry, nephelometry, FACS, etc., which are known to those skilled in the art.

[0105] The relative abundance of marker genes for decidual cells and decidual senescent cells can be expressed as a ratio, such as the PLA2G2A / DIO2 ratio. The fold change in this ratio provides information about the levels of each of these marker genes and can be used in the methods described herein. An increase in the level of a marker gene for decidual cells, and optionally a decrease in the level of a marker gene for decidual senescent cells, will result in an increase in the ratio. A decrease in the level of a marker gene for decidual cells, and optionally an increase in the level of a marker gene for decidual senescent cells, will result in a decrease in the ratio.

[0106] Methods for monitoring or evaluating treatment effectiveness

[0107] The present invention also provides a method for monitoring or evaluating the effectiveness of a treatment to reduce the risk (or likelihood or probability) of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene in decidual cells and at least one marker gene in decidual senescent cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effectiveness of the treatment, wherein the at least one marker gene in the decidual cells comprises PLA2G2A. As described in the previous section, the relationship between the levels of marker genes in decidual cells and decidual senescent cells enables determination of the risk of pregnancy loss or implantation failure, thereby determining whether the treatment is effectively reducing the risk. Treatments that can be used to reduce the risk are further described below.

[0108] The marker genes and samples that can be used in the methods of monitoring or evaluating the effects of treatment can be any of the marker genes and samples described in the preceding sections.

[0109] Monitoring or evaluating the effect of treatment to reduce the risk of pregnancy loss or embryo implantation failure includes determining whether the individual is responding to or has responded to treatment, determining the nature of the response, determining the extent of the response, and determining whether the individual continues to respond to treatment in the same manner over time. In some cases, the individual is determined to be responding to treatment or to have a positive response. Responsiveness or a positive response to treatment means that as a result of treatment, the individual is expected to obtain a benefit or a sufficient degree of benefit. For example, the individual can have or expect a successful conception, or the individual can have an improved prognosis. Non-responsiveness or a negative response to treatment means that the individual is not expected to obtain a benefit or a sufficient degree of benefit from receiving treatment.

[0110] Compared to a reference sample or level, the level of a marker gene for decidual cells (e.g., PLA2G2A) that has increased or is increasing in a sample can indicate a positive response to treatment. Alternatively, the level of a marker gene for decidual senescent cells (e.g., DIO2) that has decreased or is decreasing compared to a reference sample or level can indicate a positive response to treatment. In one example, the level of a marker gene for decidual cells (e.g., PLA2G2A) that has increased or is increasing in a sample and the level of a marker gene for decidual senescent cells (e.g., DIO2) that has decreased or is decreasing compared to a reference sample or level can together indicate a positive response to treatment. The relative abundance of marker genes for decidual cells and decidual senescent cells can be expressed as a ratio, such as a PLA2G2A / DIO2 ratio. A fold change in this ratio can indicate whether the treatment is effective. For example, an increase in the level of a marker gene for decidual cells, and optionally, a decrease in the level of a marker gene for decidual senescent cells, will result in an increased ratio, indicating a positive response to treatment resulting from reduced decidual senescence.

[0111] Compared to a reference sample or level, the level of a marker gene (e.g., PLA2G2A) for decidual cells that has decreased, is decreasing, or is unchanged in the sample can indicate a negative response to treatment. Alternatively, the level of a marker gene (e.g., DIO2) for decidual senescent cells that has increased, is increasing, or is unchanged in the sample can indicate a negative response to treatment compared to a reference sample or level. In one example, the level of a marker gene (e.g., PLA2G2A) for decidual cells that has decreased, is decreasing, or is unchanged in the sample, and the level of a marker gene (e.g., DIO2) for decidual senescent cells that has increased, is increasing, or is unchanged in the sample can together indicate a negative response to treatment compared to a reference sample or level. The ratio of marker genes for decidual cells to decidual senescent cells may decrease due to a decrease in the level of marker genes for decidual cells and, optionally, an increase in the level of marker genes for decidual senescent cells, indicating a negative response to treatment.

[0112] In one example, a method for monitoring or evaluating the effectiveness of a treatment for reducing the risk of pregnancy loss or embryo implantation failure in an individual further comprises detecting and / or quantifying the level of uNK cells or the level of at least one marker gene for uNK cells in a sample. For example, the detection and / or quantification of the level of uNK cells based on marker genes can be as described in the preceding section.

[0113] Increased or increasing levels of uNK cells or uNK cell gene markers in a sample compared to a reference sample can indicate a positive response to treatment, whereas decreased or decreasing levels of uNK cells or uNK cell gene markers in a sample compared to a reference sample can indicate a negative response to treatment.

[0114] The above-mentioned treatment methods of the present invention may further comprise detecting and / or quantifying genes that allow identification of the phase, point or day in the menstrual cycle, as described in the previous section.

[0115] A control sample or reference sample or level can be selected according to any of the above criteria. In a method for monitoring or evaluating the therapeutic effect of reducing the risk of pregnancy loss or embryo implantation failure in an individual, the level of the marker gene (e.g., PLA2G2A and DIO2) at the first time point before treatment can be compared with the level of the marker gene (e.g., PLA2G2A and DIO2) at a later time point during or after treatment. The level of the marker gene (e.g., PLA2G2A and DIO2) during treatment can also be compared with the level of the marker gene (e.g., PLA2G2A and DIO2) at a later time point during or after treatment. In some cases, the level of the marker gene can be determined monthly, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, every twelve months, or at any other suitable time interval determined by a licensed physician.

[0116] In related aspects of the above methods, the present invention also provides a method for preventing or reducing the risk of pregnancy loss or implantation failure in an individual, wherein the at least one marker gene of decidual cells comprises PLA2G2A, and wherein the method comprises detecting and / or quantifying the amount of the at least one marker gene of decidual cells and the at least one marker gene of decidual senescent cells in a biological sample obtained from the individual, and administering an agent or implementing a treatment regimen effective to prevent or reduce the risk of pregnancy loss or implantation failure in the individual. If the marker gene level indicates a risk of pregnancy loss or implantation failure as described above, the agent is administered or the treatment regimen is implemented.

[0117] Methods for diagnosing reproductive disorders

[0118] The present invention also provides a method for diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, thereby diagnosing the disorder, wherein the at least one marker gene for decidual cells comprises PLA2G2A. Marker genes useful in methods for diagnosing reproductive disorders are as described in the preceding sections.

[0119] Reproductive disorder can be any reproductive disorder. Reproductive disorder can be any obstacle relevant to infertility, miscarriage, any obstacle relevant to the risk of obstetric complications or to the pregnancy outcome having a negative impact. Reproductive disorder as herein described can be any obstacle comprising reducing receptivity or endometrial receptivity to embryo failure. Such obstacle can comprise embryo implantation failure, miscarriage, recurrent pregnancy loss or placental disease. In a preferred embodiment, reproductive disorder is recurrent pregnancy loss.

[0120] Diagnosis involves determining whether an individual has a reproductive disorder. Diagnosis can also include determining the specific cause of the reproductive disorder and identifying different clinical manifestations. A positive diagnosis involves determining that an individual has a disorder. A negative diagnosis involves determining that an individual does not have a disorder.

[0121] Compared to a reference sample or level, a decreased or decreasing level of a marker gene for decidual cells (e.g., PLA2G2A) in a sample can indicate a positive diagnosis. Alternatively, an increased or increasing level of a marker gene for decidual senescent cells (e.g., DIO2) can indicate a positive diagnosis compared to a reference sample or level. In one example, a decreased or decreasing level of a marker gene for decidual cells (e.g., PLA2G2A) in a sample and an increased or increasing level of a marker gene for decidual senescent cells (e.g., DIO2) can together indicate a positive diagnosis compared to a reference sample or level.

[0122] Compared to a reference sample or level, the level of a marker gene (e.g., PLA2G2A) for decidual cells that is unchanged (or similar), increased, or increasing in the sample can indicate a negative diagnosis. Alternatively, the level of a marker gene (e.g., DIO2) for decidual senescent cells that is unchanged (or similar), decreased, or decreasing in the sample can indicate a negative diagnosis. In one example, the level of a marker gene (e.g., PLA2G2A) for decidual cells that is unchanged (or similar), increased, or increasing in the sample, and the level of a marker gene (e.g., DIO2) for decidual senescent cells that is unchanged (or similar), decreased, or decreasing in the sample, compared to a reference sample or level, can together indicate a negative diagnosis.

[0123] In one example, the method of diagnosing a reproductive disorder in an individual further comprises detecting and / or quantifying the level of uterine natural killer (uNK) cells, for example based on the level of at least one marker gene for uNK cells in the sample. The uNK cell marker genes that can be used in the diagnostic method can be as described in the preceding section.

[0124] A decreased or decreasing level of uNK cells in the sample compared to the reference sample indicates a positive diagnosis. An unchanged (or similar), increased, or increasing level of uNK cells compared to the reference sample indicates a negative diagnosis.

[0125] The diagnostic method of the present invention may further comprise the detection and / or quantification of genes that allow identification of the day in the menstrual cycle, as described in the previous section.

[0126] A control sample or reference sample or level can be provided according to the standards described above and can represent levels from an individual or individuals known to have a reproductive disorder or known not to have any reproductive disorder.

[0127] Treatment

[0128] According to the present invention, methods for treating a reproductive disorder in an individual are also provided. The methods include diagnosing the reproductive disorder according to the methods described in the preceding sections, and administering an agent or implementing a treatment regimen to effectively treat the reproductive disorder in an individual diagnosed with a positive diagnosis. Also described are agents for use in methods of treating a reproductive disorder in an individual, wherein the reproductive disorder is diagnosed according to the methods described in the preceding sections. Also described are uses of the agents for preparing a medicament for treating a reproductive disorder. In some embodiments, the individual has increased levels of at least one marker gene for decidual senescent cells (e.g., DIO2). In some embodiments, the individual has decreased levels of at least one marker gene for decidual cells (e.g., PLA2G2A). The term "treating" includes alleviating or preventing the development or progression of a disorder, as well as reducing or eliminating an existing disorder or its symptoms. For example, an individual can be considered treated if marker levels are altered, resulting in a negative diagnosis. For example, the relative abundance of marker genes for decidual cells and decidual senescent cells can be expressed as a ratio, such as the PLA2G2A / DIO2 ratio. A fold change in this ratio can indicate whether the treatment is effective. For example, an increase in the level of a marker gene for decidual cells, and optionally, a decrease in the level of a marker gene for decidual senescent cells, will result in an increased ratio, indicating a positive response to treatment by attenuating decidual senescence. The ratio of marker genes for decidual cells to decidual senescent cells may also be decreased, due to a decrease in the level of a marker gene for decidual cells and optionally an increase in the level of a marker gene for decidual senescent cells, indicating a negative response to treatment.

[0129] The medicament or treatment regimen that can be applied or implemented can be any medicament or treatment regimen known to be effective for treating reproductive disorders. The medicament or treatment regimen can be any medicament or treatment regimen that can increase the level of decidual cells and / or uNK cells in an individual, and / or the medicament or treatment regimen of the level of decidual senescent cells reduced in an individual. Suitable medicaments or treatment regimens can include but are not limited to endometrial scraping, dipeptidyl peptidase IV (DPP4) inhibitors (usually gliptins, such as sitagliptin (sitagliptin)) and anti-aging drugs (senolytic drugs) (e.g., dasatinib, quercetin). Examples of DPP4 inhibitors include, for example, vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin, evogliptin, omarigliptin, teneligliptin, and are described, for example, in Deacon CF & Lebovitz HE, Diabetes Obes Metab., 2016; 18(4): 333-47. As determined based on the diagnostic methods previously described, agents or treatment regimens can target different types of decidual disorders. For example, endometrial scraping can be used to treat decidual failure. Senolytic drugs can be used to treat age-related reproductive disorders. Senolytic drugs (senolytic drugs or senolytics) are drugs that can target cellular senescence in order to delay, prevent, alleviate or reverse age-related disorders. The above-described agents and treatment regimens are also described for use in the above-described methods of reducing the risk of or preventing pregnancy loss or embryo implantation failure.

[0130] In a preferred aspect, the agent is a DPP4 inhibitor or antagonist. DPP4 is a known marker of glandular differentiation in the mid-luteal phase and is a ubiquitous aminopeptidase expressed as a cell surface-bound protein and a soluble form (59, 60). DPP4 is also a widely used marker gene for endometrial receptivity (61). Stromal cell-derived factor 1α (SDF-1), also known as CXC motif chemokine ligand 12 (CXCL12), is a potent chemokine that mediates BMDC mobilization and homing to the endometrium in response to tissue damage and elevated estradiol levels (62, 63). However, SDF-1 is inactivated by DPP4 proteolysis. The present inventors have found that oral antidiabetic drugs DPP4 inhibitors (gliptins), which are commonly used to treat type 2 diabetes (64), can be used to reduce excessive decidual aging in RPL patients by increasing endometrial stem cells or inhibiting the expression of marker genes for senescent decidual cells (e.g., DIO2).

[0131] A DPP4 inhibitor or antagonist can be any agent that inhibits or antagonizes DPP4 expression or activity by any means. The agent can inhibit or antagonize the inactivation of SDF-1 by DPP4. Such an agent can be a small molecule, a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA) or a small hairpin RNA (shRNA) or any other suitable inhibitor that achieves the above functions. The agent can be a polynucleotide that encodes a molecule that inhibits or antagonizes DPP4, or can be a polynucleotide, an oligonucleotide, an antisense RNA, a siRNA or a shRNA that inhibits the expression of DPP4, typically comprising a sequence complementary to DPP4 mRNA and specifically hybridizing therewith. When the oligonucleotide hybridizes with the target sequence with preferential or high affinity, but does not hybridize substantially, does not hybridize or hybridizes only with low affinity to other sequences, the oligonucleotide "specifically hybridizes" to the target sequence. More preferably, the oligonucleotide hybridizes with a T of at least 5°C, at least 10°C, at least 20°C, at least 30°C or at least 40°C. m Hybridizes to the target sequence with a T greater than that of other nucleic acids. mConditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: alaboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995)). Hybridization conditions may be stringent conditions as described in the art.

[0132] The pharmaceutical agent can be an antibody that specifically binds to the DPP4 protein or another protein to indirectly inhibit DPP4 function. An antibody "specifically binds" to a protein when it binds preferentially or with high affinity to the protein, but does not substantially bind, does not bind, or binds only with low affinity to other proteins. For example, an antibody "specifically binds" to a target molecule when it binds preferentially or with high affinity to the target molecule, but does not substantially bind, does not bind, or binds only with low affinity to other human proteins.

[0133] If the antibody is present at 1×10 -7 M or less, more preferably 5×10 -8 M or less, more preferably 1×10 -8 M or less or more preferably 5×10 -9 If the antibody binds with a Kd of 1×10 -6 M or more, more preferably 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, even more preferably 1×10 -2 If the Kd is above 5 M, the antibody binds with low affinity.

[0134] The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a bispecific antibody, a CDR-grafted antibody, or a humanized antibody. The antibody can be a complete immunoglobulin molecule or a fragment thereof, such as a Fab, F(ab')2, or Fv fragment.

[0135] In a preferred embodiment, the medicament used in the treatment method can be a gliptin, for example, including sitagliptin, vildagliptin, saxagliptin, linagliptin, gemagliptin, anagliptin, tegliptin, alogliptin, trelagliptin, alogliptin, ipagliptin, gosogliptin or dutogliptin. Preferably, the gliptin is sitagliptin.

[0136] In some embodiments, treatment may include a step of detecting an increase in the level of a marker gene (e.g., DIO2) for decidual senescent cells. When an increase in DIO2 is detected, the agent used is preferably a DPP4 inhibitor, typically a gliptin, and more preferably sitagliptin.

[0137] The specific route, dosage and method of administration of the therapeutic agents described herein can be routinely determined by a medical practitioner. The agents used in the methods of treatment described herein can be formulated in pharmaceutical compositions. In addition to the therapeutically active ingredient, these compositions can contain pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent can be, for example, an isotonic solution.

[0138] The dosage can be determined according to various parameters, in particular the agent used; the age, weight and condition of the patient to be treated; the route of administration; and the desired regimen. Similarly, the physician will be able to determine the route of administration and dosage required for any particular patient.

[0139] Medicament can be administered to the patient by any suitable means.Medicament can be administered by enteral or parenteral route, for example, by oral, oral cavity, anus, lung, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraarticular, local or other suitable route of administration.For example, in the case where medicament is a DPP4 inhibitor (such as sitagliptin), it is preferably administered orally.

[0140] The daily dose of gliptins, such as sitagliptin, administered to a subject (e.g., a human) can be from about 50 mg / day to about 2000 mg / day, such as from about 50 mg / day to about 1500 mg / day, from about 50 mg / day to about 100 mg / day, from about 75 mg / day to about 150 mg / day, from about 100 mg / day to about 1500 mg / day, from about 100 mg / day to about 1200 mg / day, from about 100 mg / day to about 175 mg / day, from about 150 mg / day to about 300 mg / day, from about 2 ...50 mg / day to about 1500 mg / day, from about 100 mg / day to about 175 mg / day, from about 150 mg / day to about 300 mg / day, from about 200 mg / day to about 1500 mg / day, from about 100 mg / day to about 1200 mg / day, from about 100 mg / day to about 175 mg / day, from about 150 mg / day to about 300 mg / day, from about 200 mg / day to about 1500 mg / day. Preferably, the typical daily dose of sitagliptin is about 100 mg / day or at least about 100 mg / day.

[0141] Administration can be a single dose or multiple doses. Multiple doses can be administered via the same or different routes and to the same or different positions. Alternatively, the dosage can be by sustained-release formulations, in which case less frequent administration is required. Dosage and frequency can vary according to the half-life of the agent in the patient and the required duration of treatment. The dosage as described above can be administered once a day, or can be divided into two doses. The medicament can be administered for more than one menstrual cycle, for example, at least two or at least three consecutive menstrual cycles. For example, 100 mg of sitagliptin capsules can be taken orally once a day for 2 or 3 consecutive menstrual cycles.

[0142] Treatment methods for medical use may include the use of additional agents known to be effective in treating reproductive disorders in individuals. For example, progesterone and / or progestogens may be additionally administered.

[0143] Also provided herein are methods for preventing pregnancy loss or embryo implantation failure, comprising detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from an individual, thereby assessing the individual as being at risk for pregnancy loss or embryo implantation failure, and administering a pharmaceutical agent or implementing a treatment regimen to effectively prevent pregnancy loss or embryo implantation failure, wherein the at least one marker gene for decidual cells comprises PLA2G2A. The pharmaceutical agent or treatment regimen can be any of the pharmaceutical agents or treatment regimens described above, preferably, a gliptin, such as sitagliptin, is administered.

[0144] Also provided are methods for assessing readiness for conception or successful embryo implantation. Such methods further involve detecting and / or quantifying the amount of at least one marker gene in decidual cells and at least one marker gene in decidual senescent cells in a biological sample obtained from an individual, thereby assessing readiness for conception or embryo implantation, wherein the at least one marker gene in the decidual cells comprises PLA2G2A. Marker genes and detection methods useful in the above methods are as described in the preceding sections.

[0145] Methods of selecting patients for treatment

[0146] The present invention describes a method for selecting a patient for treatment to reduce the risk (or likelihood or probability) of embryo implantation failure or pregnancy loss in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells in a biological sample obtained from an individual, and thereby selecting a patient for treatment based on the level of the marker gene to reduce the risk of pregnancy loss or embryo implantation failure, wherein at least one marker gene of the decidual cells comprises PLA2G2A. The method can include detecting and / or quantifying the level of uNK cells in the sample or uNK cell gene markers in the sample. The marker genes that can be used in the method for selecting a patient are those described in the previous section. Treatment can use any therapeutic regimen or agent as described above.

[0147] Individuals in whom an increased level of at least one marker gene for decidual senescent cells (e.g., DIO2) is detected can be selected as patients receiving treatment. Individuals in whom a decreased level of at least one marker gene for decidual cells (e.g., PLA2G2A) is detected can also be selected as patients receiving treatment. The selected patients are preferably treated with a DPP4 inhibitor. Preferably, the marker gene for decidual senescent cells is DIO2, and the selected patients are treated with sitagliptin.

[0148] The methods of the present invention may further comprise detecting and / or quantifying genes that allow identification of days in the menstrual cycle, as described in the previous section.

[0149] The relationship between the levels of marker genes in decidual cells, decidual senescent cells, and uNK cells can enable the identification of specific defects in the decidual pathway. It can then be determined whether a patient might benefit from a specific type of treatment to reduce the risk of embryo implantation failure or miscarriage.

[0150] Methods for stratifying patients

[0151] The present invention also provides a method for stratifying patients into different groups, for example for clinical research. The method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from an individual, wherein the at least one marker gene for the decidual cells comprises PLA2G2A. The method may include detecting and / or quantifying the level of uNK cells in the sample, for example based on the level of uNK cell gene markers in the sample. The marker genes that can be used in the method for stratifying patients can be any marker gene as described in the preceding section.

[0152] The methods of the present invention may further comprise detecting and / or quantifying genes that allow identification of days in the menstrual cycle, as described in the previous section.

[0153] The relationship between the levels of marker genes in decidual cells, decidual senescent cells, and uNK cells can enable the identification of specific defects in the decidual pathway. These patients with different patterns or levels of markers can then be grouped accordingly for clinical studies.

[0154] Reagent test kit

[0155] The present invention also provides a kit that can be used in any of the methods of the present invention. The kit can include a device (e.g., a reagent) for detecting and / or quantifying at the nucleic acid or protein level at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample from an individual, wherein the at least one marker gene for decidual cells comprises PLA2G2A. The kit thus includes a reagent for detecting and / or quantifying PLA2G2A. The at least one marker gene for decidual senescent cells can comprise DIO2. The at least one marker gene for decidual senescent cells can be selected from DIO2, CLU, and IGFBP1. The kit can also include a device for detecting and / or quantifying levels of uNK cells or uNK cell markers. The marker genes that can be detected using the kit can be any of those described in the preceding sections. The kit can further include a device for detecting and / or quantifying genes that allow for identification of dates in the menstrual cycle, as described in the preceding sections. Preferably, the genes that allow for identification of the timing of dates in the menstrual cycle comprise, consist of, or consist essentially of GPX3 and SLC15A2.

[0156] The only reagent for detecting and / or quantifying a marker gene contained in the kit may be a reagent for detecting the aforementioned marker gene.

[0157] The kit may also include instructions for using the kit according to the methods of the present invention. The kit may also include details regarding which individuals can use the method. The kit may also be provided with a device for obtaining an endometrial biopsy sample. The kit may also include a test request form containing detailed information to be sent to an analyst. The kit may also include devices for measuring other laboratory or clinical parameters, and / or a container for containing a biological sample isolated from a subject.

[0158] The kit may further comprise one or more other reagents or instruments that enable the method to be implemented. Such reagents or instruments may include one or more of the following: a suitable buffer (aqueous solution), a calibration curve standard, a developing reagent, an enzyme, a label, a reaction surface, a device for detection, a control sample, a standard, instructions, interpretive information, a device for isolating the relevant biomarker from the sample, a device for obtaining a sample from an individual (such as a container or an instrument comprising a needle), or a support comprising a well on which a quantitative reaction can be performed.

[0159] In one example, the kit can include a cryotube and an RNA stabilization solution.

[0160] In one embodiment, the use of the above-mentioned detection kit for assessing pregnancy loss or embryo implantation failure or for diagnosing reproductive disorders is described. The use may include the steps described above for the method for detecting marker genes of the present invention. In a preferred embodiment, the kit is used to assess the risk of recurrent pregnancy loss or diagnose recurrent pregnancy loss.

[0161] Example

[0162] Materials and methods

[0163] Ethical approval and sample collection

[0164] This study was approved by the NHS National Research Ethics - Hammersmith and Queen Charlotte's & Chelsea Research Ethics Committee (1997 / 5065). All samples were obtained with written informed consent and in accordance with the guidelines of the Declaration of Helsinki (2000). Human endometrial biopsies were obtained from women attending the Implantation Clinic, a dedicated research clinic located at University Hospitals Coventry and Warwickshire (UHCW) National Health Service Trust. Surplus tissue from endometrial biopsies obtained for diagnostic purposes at the Implantation Research Clinic was used in this study. Endometrial biopsies were collected using the Wallach Endocell ELISA kit during the luteal phase of the ovulatory, non-hormonally stimulated menstrual cycle, with the preovulatory LH surge as the time reference. TM An endometrial sample is obtained using an endometrial biopsy device. Transvaginal ultrasound scans are performed to exclude significant uterine pathology before biopsy.

[0165] Drop-Seq analysis of timed endometrial biopsies

[0166] Six LH-timed endometrial biopsies were processed as described elsewhere (Lucas et al., 2020). After tissue digestion, red blood cells were removed from the effluent by Ficoll density gradient centrifugation. Single-cell fractions were then subjected to Drop-Seq analysis.

[0167] Reverse transcription quantitative PCR (RT-qPCR)

[0168] RNA was extracted from endometrial biopsies that had been placed in the RNA Later at the clinic (<1 min after collection) using the RNeasy plus Universal mini kit (QIAGEN) according to the manufacturer's instructions. Reverse transcription was performed from 1 μg of RNA using the Quantitect Reverse Transcription Kit (QIAGEN), and cDNA was diluted to 10 ng / μl equivalent before use in qPCR. Amplification was performed in 10 μl reactions using a 2× Quantifast SYBR Green RT-PCR Kit (QIAGEN) on a QuantStudio 5 (ThermoFisher), with 300 nM each of the forward and reverse primers. Primer sequences are as follows: DIO2 forward: 5′-ACT CGG TCA TTC TGC TCA A-3′, DIO2 reverse: 5′-TTC CAG ACGCAG CGC AGT-3′, PLA2G2A forward: 5′AAA GGA AGC CGC ACT CAG TT-3′, PLA2G2A reverse: 5′-TTTCCA GGG AAG AGG GGA C-3′. Percentile dCt values were calculated using R v3.5 software.

[0169] Multiplex single-molecule in situ hybridization (RNAScope)

[0170] Formalin-fixed paraffin-embedded (FFPE) samples were cut into 5 μm sections. RNA in situ hybridization was performed according to the manufacturer's instructions using a probe for PLA2G2A (581101-C2) and a probe for DIO2 (562211). 2.5HD Duplex Reagent Kit (ACD, California, USA). After hybridization and amplification, slides were counterstained with 50% hematoxylin. Images were acquired using a Mirax Midi slide scanner with a 20× objective and opened in Panoramic Viewer v1.15.4 (3DHISTECH Ltd) for analysis.

[0171] Spatial transcriptomics

[0172] Endometrial biopsies were fixed overnight in 10% neutral buffered formalin at 4°C and then embedded in paraffin using a Shandon Excelsior ES tissue processor (ThermoFisher) with Surgipath Formula 'R' paraffin. Four endometrial biopsies were selected based on morphology, PLA2G2A / DIO2 expression, and RNA integrity (DV.200>50). 5 μm sections were prepared, dewaxed, and stained with hematoxylin and eosin according to the operating procedures (10x Genomics). Spatial gene expression slides and reagent kits were used according to the manufacturer's instructions. Each capture area (6.5×6.5 mm 2 ) contains 5,000 barcoded spots with a diameter of 55 μm (100 μm between spot centers), providing an average resolution of 1 to 10 cells. The eluted libraries were analyzed using an Agilent Bioanalyzer High Sensitivity DNA chip to assess quality and determine library size. Library dilution and denaturation were performed according to standard Illumina protocols and sequenced using the NextSeq 500 / 550 High Output Kit v2.5 (150 cycles). Sequencing was performed with the recommended 10X protocol (read 1: 28 cycles; i7 index reads: 10 cycles; i5 index reads: 10 cycles; and read 2: 91 cycles), generating 21 million to 50 million sequencing reads. Reads were processed using Spaceranger software v1.3.0 with reference genome data refdata-gex-GRCh38-2020-A and analyzed in R v4.1.3 using Seurat v4.0.4.

[0173] Bulk RNA-Sequencing

[0174] Total RNA was extracted from endometrial biopsies using the RNeasy plus Universal mini kit (QIAGEN) according to the manufacturer's instructions. Library dilution and denaturation were performed according to standard Illumina protocols and sequencing was performed using the NextSeq 500 / 550 High Output Kit v2.5 (75 cycles). Reads were mapped to the GRCh38 human genome assembly and gencode v38 annotations using STAR v2.7.9. Overlapping gene regions were counted using HTSeq v0.6.1 with the "intersection-nonempty" option. Differential gene expression analysis was performed using DESeq2 v1.34.0 in R.

[0175] SIMPLANT clinical trial data

[0176] Details of the SIMPLANT clinical trial's methods and management can be found in Tewary et al., 2020. Briefly, a double-blind, randomized, placebo-controlled feasibility trial was conducted in women aged 18 to 42 years with a history of three or more miscarriages. Thirty-eight subjects were randomly assigned to receive three consecutive cycles of the DPP-4 inhibitor sitagliptin (100 mg daily) or an equivalent placebo capsule. Exploratory studies were performed on tissue samples.

[0177] result

[0178] 1. Discovery of new biomarkers of anti-inflammatory decidual cells.

[0179] Using high-throughput single-cell droplet barcoding technology, transcriptome changes in the decidualization pathway in vitro were analyzed to define the differentiation characteristics of decidual subpopulations. Single-cell RNA sequencing analysis of luteal phase endometrial biopsies initially identified two putative biomarkers, SCARA5 and DIO2, as selective markers for decidual cells and stressed / senescent cells, respectively (Lucas et al., 2020; WO 2021 / 032973).

[0180] Further bulk RNA sequencing (RNA-seq) of paired biopsies, obtained in the same patient but at different cycles, identified the biomarker gene PLA2G2A, which encodes phospholipase A2 group IIA ( Figure 2 A and 2B). Like SCARA5, PLA2G2A is also a stromal cell-specific biomarker gene for progesterone-dependent decidual cells, but its expression level has a larger dynamic range, making it a significantly more sensitive biomarker. Both DIO2 and PLA2G2A are highly enriched in the endometrial stroma ( Figure 2 C), although they show distinct temporal regulation throughout the menstrual cycle ( Figure 2 D). We generated reference ranges for expression of these two genes in the periimplantation endometrium by RTQ-PCR analysis of 822 biopsy samples obtained 6 to 11 days after the preovulatory luteinizing hormone surge (LH+6 to LH+11) as determined by over-the-counter home ovulation test kits ( Figure 2 E). Percentiles were used to compare the relative expression of biomarkers in endometrial samples obtained on different days of the menstrual cycle.

[0181] Multiplex single-molecule in situ hybridization The spatial organization of PLA2G2A and DIO2 expressing stromal subpopulations was elucidated by using Visium spatial transcriptomics (10xGenomics). Spatial analysis confirmed that DIO2 and PLA2G2A label distinct stromal subpopulations in the peri-implantation endometrium. Figure 3Furthermore, while DIO2-positive cells are enriched in the area adjacent to the luminal epithelium (which lines the uterine cavity), PLA2G2A-positive cells are found deep within the tissue. Therefore, quantification of DIO2 and PLA2G2A transcript levels in endometrial samples provides information about the spatial organization of the tissue during the peri-implantation window.

[0182] 2. Diagnostic and prognostic potential of the standardized PLA2G2A / DIO2 expression ratio in luteal phase endometrial biopsies in recurrent pregnancy loss

[0183] We analyzed the PLA2G2A / DIO2 ratio (expressed as percentiles of the ratio) in 854 LH-timed endometrial biopsies from women with a history of 0 to 18 prior miscarriages. The lower the ratio of these marker genes, the greater the relative excess of stress / senescent cells versus anti-inflammatory decidual cells in the sample, and vice versa. Figure 4 As shown in Figure A, in this sample group, the median PLA2G2A / DIO2 ratio decreased progressively with increasing number of previous miscarriages. With each additional loss, the frequency of samples with a ratio below the 25th percentile (lower quartile) decreased, while the frequency of samples with a ratio above the 75th percentile (upper quartile) increased ( Figure 4 B). A cutoff of PLA2G2A / DIO2 ratio < 15th percentile maximized the separation of samples from women with no previous pregnancy loss and women with more than 6 miscarriages ( Figure 4 C). Importantly, there were no significant differences in maternal age or timing of the biopsy relative to the preovulatory LH surge between samples below or above the 15th percentile cutoff ratio ( Figure 4 D). However, patients with a ratio below the 5th percentile cutoff had a significantly higher body mass index (BMI) (P value < 0.001), which is a well-documented risk factor for recurrent miscarriage / recurrent pregnancy loss (Quenby et al., 2021).

[0184] We also analyzed 217 endometrial biopsies obtained before subsequent pregnancies. There were no restrictions on the time interval between the biopsy date and the start of pregnancy. Figure 5 As shown in A, patients with recurrent miscarriage who had a low PLA2G2A / DIO2 ratio were significantly more likely to have another miscarriage (<20th percentile). In contrast, patients with a high PLA2G2A / DIO2 ratio (>60th percentile) were less likely to have another miscarriage. Importantly, there were no significant differences in maternal age, body mass index (BMI), and uNK cell levels between patients who had a live birth or pregnancy loss after endometrial biopsy (P>0.05) ( Figure 5 B).

[0185] 3. Therapeutic intervention: Sitagliptin treatment improves the PLA2G2A / DIO2 expression ratio in recurrent pregnancy loss.

[0186] We previously reported that sitagliptin, a dipeptidyl peptidase IV (DPP4) inhibitor used in the management of type 2 diabetes, increases the recruitment of bone marrow-derived MSCs when administered over 3 menstrual cycles (Tewary et al., 2020). We investigated the effects of sitagliptin versus placebo on the endometrial PLA2G2A / DIO2 ratio before and after the trial using endometrial samples obtained during this double-blind, placebo-controlled, randomized pilot trial. Figure 6 As shown, patients receiving sitagliptin significantly improved their endometrial PLA2G2A / DIO2 ratio (P=0.0052). In contrast, the endometrial PLA2G2A / DIO2 ratio did not differ significantly before and after the trial in the placebo group (P>0.05).

[0187] References

[0188] 1 Rai, R. & Regan, L. Recurrent miscarriage. Lancet 368, 601-611, doi:10.1016 / S0140-6736(06)69204-0(2006).

[0189] 2 Hardy, K., Hardy, PJ, Jacobs, PA, Lewallen, K. & Hassold, TJ Temporal changes in chromosome abnormalities in human spontaneous abortions: Results of 40 years of analysis. Am J Med Genet A 170, 2671-2680, doi:10.1002 / ajmg.a.37795(2016).

[0190] 3 Hassold, T. et al. A cytogenetic study of 1000 spontaneoabortions. Ann Hum Genet 44, 151-178 (1980).

[0191] 4 Ogasawara,M.,Aoki,K.,Okada,S.&Suzumori,K.Embryonic karyotype ofabortuses in relation to the number of previous miscarriages.Fertility andsterility 73,300-304(2000).

[0192] 5 Stephenson,M.D.,Awartani,K.A.&Robinson,W.P.Cytogenetic analysis ofmiscarriages from couples with recurrent miscarriage:a case-control study.HumReprod 17,446-451(2002).

[0193] 6 Sullivan,A.E.,Silver,R.M.,LaCoursiere,D.Y.,Porter,T.F.&Branch,D.W.Recurrent fetal aneuploidy and recurrent miscarriage.Obstet Gynecol 104,784-788,doi:10.1097 / 01.AOG.0000137832.86727.e2(2004).

[0194] 7 Carp,H.et al.Karyotype of the abortus in recurrentmiscarriage.Fertil Steril 75,678-682(2001).

[0195] 8 Robberecht,C.et al.Cytogenetic and morphological analysis of earlyproducts of conception following hystero-embryoscopy from couples withrecurrent pregnancy loss.Prenat Diagn 32,933-942,doi:10.1002 / pd.3936(2012).

[0196] 9 ESHRE.Recurrent Pregnancy Loss:A Guideline of the European Societyof Human Reproduction and Embryology.(2017).

[0197] 10 Practice Committee of the American Society for Reproductive,M.Evaluation and treatment of recurrent pregnancy loss.Fertility andSterility 5,1103-1111(2012).

[0198] 11 Cha,J.,Sun,X.&Dey,S.K.Mechanisms of implantation:strategies forsuccessful pregnancy.Nature medicine 18,1754-1767,doi:10.1038 / nm.3012(2012).

[0199] 12 Moraes,J.G.N.et al.Uterine influences on conceptus development infertility-classified animals.Proc Natl Acad Sci U S A 115,E1749-E1758,doi:10.1073 / pnas.1721191115(2018).

[0200] 13 Salker,M.S.et al.Disordered IL-33 / ST2 activation in decidualizingstromal cells prolongs uterine receptivity in women with recurrent pregnancyloss.PLoS One 7,e52252,doi:10.1371 / journal.pone.0052252(2012).

[0201] 14 Gellersen,B.&Brosens,J.J.Cyclic decidualization of the humanendometrium in reproductive health and failure.Endocrine reviews 35,851-905,doi:10.1210 / er.2014-1045(2014).

[0202] 15 Brighton,P.J.et al.Clearance of senescent decidual cells byuterine natural killer cells in cycling human endometrium.eLife 6,doi:10.7554 / eLife.31274(2017).

[0203] 16 Nancy,P.et al.Chemokine gene silencing in decidual stromal cellslimits T cell access to the maternal-fetal interface.Science 336,1317-1321,doi:10.1126 / science.1220030(2012).

[0204] 17 Macosko,E.Z.et al.Highly Parallel Genome-wide Expression Profilingof Individual Cells Using Nanoliter Droplets.Cell 161,1202-1214,doi:10.1016 / j.cell.2015.05.002(2015).

[0205] 18 Lucas,E.S.et al.Loss of Endometrial Plasticity in RecurrentPregnancy Loss.Stem Cells 34,346-356,doi:10.1002 / stem.2222(2016).

[0206] 19 Lucas,E.S.,Vrljicak,P.,Muter,J.,Diniz-Da-Costa,M.M.,Brighton,P.J.,Kong,C.S.,Lipecki,J.,Fishwick,K.J.,Odendaal,J.,Ewington,L.J.,Quenby,S.,Ott,S.&Brosens,J.J.Recurrent pregnancy loss is associated with a pro-senescentdecidual response during the peri-implantation window.Commun Biol,3,37.(2020)

[0207] 20 Tewary,S.,Lucas,E.S.,Fujihara,R.,Kimani,P.K.,Polanco,A.,Brighton,P.J.,Muter,J.,Fishwick,K.J.,Da Costa,M.,Ewington,L.J.,Lacey,L.,Takeda,S.,Brosens,J.J.&Quenby,S.Impact of sitagliptin on endometrial mesenchymal stem-like progenitor cells:A randomised,double-blind placebo-controlledfeasibility trial.EBioMedicine,51,102597.(2020)

[0208] 21 Magnus,M.C.,Wilcox,A.J.,Morken,N.-H.,Weinberg,C.R.& S.E.Roleof maternal age and pregnancy history in risk of miscarriage:Prospectiveregister based study.bmj,364.(2019)

[0209] 22 Brosens ,JJ ,Salker ,MS ,Teklenburg ,G ,Nautiyal ,J ,Salter ,S ,Lucas ,ES ,Steel ,JH ,Christian ,M ,Chan ,YW ,Boomsma ,CM ,Moore ,JD ,H artshorne, GM, Sucurovic, S., Mulac-Jericevic, B., Heijnen, CJ, Quenby, S., Koerkamp, MJ, Holstege, FC, Shmygol, A. & Macklon, NSUterine selection of human embryos and implantation.Sci Rep,4,3894.(2014).

[0210] 23 Ewington,LJ,Tewary,S.&Brosens,JJNew insights into themechanisms underlying recurrent pregnancy loss.J Obstet Gynaecol Res 45,258-265,doi:10.1111 / jog.13837(2019).

[0211] 24 Brosens ,JJ ,Bennett ,PR ,Abrahams ,VM ,Ramhorst ,R ,Coomarasamy ,A ,Quenby ,S ,Lucas ,ES&Mccoy ,RCMaternal selection of human embryos inearly gestation:Insights from recurrent miscarriage .Semin Cell Dev Biol.

[0212] 25 Al-Sabbagh,M.et al.NADPH oxidase-derived reactive oxygen speciesmediate decidualization of human endometrial stromal cells in response tocyclic AMP signaling.Endocrinology 152,730-740,doi:10.1210 / en.2010-0899(2011).

[0213] 26 Zhong J,Rajagopalan S.Dipeptidyl Peptidase-4 Regulation of SDF-1 / CXCR4 Axis:Implications for Cardiovascular Disease.Front Immunol.2015;6:477.27 Imai K,Maeda M,Fujiwara H,Kariya M,Takakura K,Kanzaki H,etal.Dipeptidyl peptidase IV as a differentiation marker of the humanendometrial glandular cells.Hum Reprod.1992;7(9):1189-94.

[0214] 28 Suhorutshenko M,Kukushkina V,Velthut-Meikas A,Altmae S,Peters M,Magi R,et al.Endometrial receptivity revisited:endometrial transcriptomeadjusted for tissue cellular heterogeneity.Hum Reprod.2018;33(11):2074-86.

[0215] 29 Wang X,Mamillapalli R,Mutlu L,Du H,Taylor HS.Chemoattraction ofbone marrow-derived stem cells towards human endometrial stromal cells ismediated by estradiol regulated CXCL12 and CXCR4 expression.Stem CellRes.2015;15(1):14-22.

[0216] 30 Yi KW,Mamillapalli R,Sahin C,Song J,Tal R,Taylor HS.Bone marrow-derived cells or C-X-C motif chemokine 12(CXCL12)treatment improve thinendometrium in a mouse model.Biol Reprod.2018.

[0217] 31 Deacon CF.A review of dipeptidyl peptidase-4 inhibitors.Hot topicsfrom randomized controlled trials.Diabetes Obes Metab.2018;20 Suppl 1:34-46.

[0218] 32 Quenby,S.,Gallos,I.D.,Dhillon-Smith,R.K.,Podesek,M.,Stephenson,M.D.,Fisher,J.,Brosens,J.J.,Brewin,J.,Ramhorst,R.,Lucas,E.S.,Mccoy,R.C.,Anderson,R.,Daher,S.,Regan,L.,Al-Memar,M.,Bourne,T.,Macintyre,D.A.,Rai,R.,Christiansen,O.B.,Sugiura-Ogasawara,M.,Odendaal,J.,Devall,A.J.,Bennett,P.R.,Petrou,S.&Coomarasamy,A.2021.Miscarriage matters:the epidemiological,physical,psychological,and economic costs of early pregnancy loss.Lancet,397,1658-1667.

[0219] 33 Erkenbrack,E.M.et al.The mammalian decidual cell evolved from acellular stress response.PLoS Biol 16,e2005594,doi:10.1371 / journal.pbio.2005594(2018).

[0220] 34 Kuroda,K.et al.Elevated periimplantation uterine natural killercell density in human endometrium is associated with impaired corticosteroidsignaling in decidualizing stromal cells.The Journal of clinicalendocrinology and metabolism 98,4429-4437,doi:10.1210 / jc.2013-1977(2013).

[0221] 35 Song,J.J.et al.Role of glutaredoxin in metabolic oxidativestress.Glutaredoxin as a sensor of oxidative stress mediated by H2O2.J BiolChem 277,46566-46575,doi:10.1074 / jbc.M206826200(2002).

[0222] 36 Zuo,R.J.et al.Crystallin alphaB acts as a molecular guard in mousedecidualization:regulation and function during early pregnancy.FEBS Lett 588,2944-2951,doi:10.1016 / j.febslet.2014.05.045(2014).

[0223] 37 Latini,F.R.et al.ABI3 ectopic expression reduces in vitro and invivo cell growth properties while inducing senescence.BMC Cancer 11,11,doi:10.1186 / 1471-2407-11-11(2011).

[0224] 38 Michishita,E.,Garces,G.,Barrett,J.C.&Horikawa,I.Upregulation ofthe KIAA1199 gene is associated with cellular mortality.Cancer Lett 239,71-77,doi:10.1016 / j.canlet.2005.07.028(2006).

[0225] 39 Petropoulou,C.,Trougakos,I.P.,Kolettas,E.,Toussaint,O.&Gonos,E.S.Clusterin / apolipoprotein J is a novel biomarker of cellular senescencethat does not affect the proliferative capacity of human diploidfibroblasts.FEBS Lett 509,287-297(2001).

[0226] 40 Trougakos,I.P.The molecular chaperone apolipoprotein J / clusterinas a sensor of oxidative stress:implications in therapeutic approaches-amini-review.Gerontology 59,514-523,doi:10.1159 / 000351207(2013).

[0227] 41 Bianco,A.C.&Kim,B.W.Deiodinases:implications of the local controlof thyroid hormone action.J Clin Invest 116,2571-2579,doi:10.1172 / JCI29812(2006).

[0228] 42 Altmae,S.et al.Meta-signature of human endometrial receptivity:ameta-analysis and validation study of transcriptomic biomarkers.Sci Rep 7,10077,doi:10.1038 / s41598-017-10098-3(2017).

[0229] 43 Vento-Tormo,R.et al.Single-cell reconstruction of the earlymaternal-fetal interface in humans.Nature 563,347-353,doi:10.1038 / s41586-018-0698-6(2018).

[0230] 44 Drury,J.A.,Tang,A.W.,Turner,M.A.&Quenby,S.A rapid,reliable methodfor uNK cell density estimation.J Reprod Immunol 97,183-185,doi:10.1016 / j.jri.2012.12.002(2013).

[0231] 45 Ramsey,E.M.,Houston,M.L.&Harris,J.W.Interactions of thetrophoblast and maternal tissues in three closely related primatespecies.American journal of obstetrics andgynecology 124,647-652(1976).

[0232] 46 Emera,D.,Romero,R.&Wagner,G.The evolution of menstruation:a newmodel for genetic assimilation:explaining molecular origins of maternalresponses to fetal invasiveness.Bioessays 34,26-35,doi:10.1002 / bies.201100099(2012).

[0233] 47 Evans,J.&Salamonsen,L.A.Inflammation,leukocytes andmenstruation.Rev Endocr Metab Disord 13,277-288,doi:10.1007 / s11154-012-9223-7(2012).

[0234] 48 Evans,J.&Salamonsen,L.A.Decidualized human endometrial stromalcells are sensors of hormone withdrawal in the menstrual inflammatorycascade.Biol Reprod 90,14,doi:10.1095 / biolreprod.113.108175(2014).

[0235] 50 O'Leary,M.A.et al.The placental mammal ancestor and the post-K-Pgradiation of placentals.Science 339,662-667,doi:10.1126 / science.1229237(2013).

[0236] 51 Marcais,A.et al.The metabolic checkpoint kinase mTOR is essentialfor IL-15 signaling during the development and activation of NK cells.NatImmunol 15,749-757,doi:10.1038 / ni.2936(2014).

[0237] 52 Mokhtar,N.M.et al.Progestin regulates chemokine(C-X-C motif)ligand14 transcript level in human endometrium.Molecular human reproduction 16,170-177,doi:10.1093 / molehr / gap100(2010).

[0238] 53 Kane,N.,Kelly,R.,Saunders,P.T.&Critchley,H.O.Proliferation ofuterine natural killer cells is induced by human chorionic gonadotropin andmediated via the mannose receptor.Endocrinology 150,2882-2888,doi:10.1210 / en.2008-1309(2009).

[0239] 54 Kao,L.C.et al.Global gene profiling in human endometrium duringthe window of implantation.Endocrinology 143,2119-2138,doi:10.1210 / endo.143.6.8885(2002).

[0240] 55 Gibson,D.A.,Greaves,E.,Critchley,H.O.&Saunders,P.T.Estrogen-dependent regulation of human uterine natural killer cells promotes vascularremodelling via secretion of CCL2.Hum Reprod 30,1290-1301,doi:10.1093 / humrep / dev067(2015).

[0241] 56 Santamaria,X.,Mas,A.,Cervello,I.,Taylor,H.&Simon,C.Uterine stemcells:from basic research to advanced cell therapies.Hum Reprod Update 24,673-693,doi:10.1093 / humupd / dmy028(2018).

[0242] 57 Murakami,K.et al.Deficiency in clonogenic endometrial mesenchymalstem cells in obese women with reproductive failure--a pilot study.PloS one8,e82582,doi:10.1371 / journal.pone.0082582(2013).

[0243] 58 Castellana,B.et al.Maternal obesity alters uterine NK activitythrough a functional KIR2DL1 / S1 imbalance.Immunol Cell Biol 96,805-819,doi:10.1111 / imcb.12041(2018).

[0244] 59 Perdu,S.et al.Maternal obesity drives functional alterations inuterine NK cells.JCI Insight 1,e85560,doi:10.1172 / jci.insight.85560(2016).

[0245] 60 Boots,C.E.,Bernardi,L.A.&Stephenson,M.D.Frequency of euploidpregnancy lossis increased in obese women with recurrent early pregnancyloss.Fertil Steril 102,455-459,doi:10.1016 / j.fertnstert.2014.05.005(2014).

[0246] 61 Leitao,B.et al.Silencing of the JNK pathway maintains progesteronereceptor activity in decidualizing human endometrial stromal cells exposed tooxidative stress signals.FASEB journal:official publication of the Federationof American Societies for Experimental Biology 24,1541-1551(2010).

[0247] 62 Leitao,B.B.,Jones,M.C.&Brosens,J.J.The SUMO E3-ligase PIAS1couples reactive oxygen species-dependent JNK activation to oxidative celldeath FASEB J 25,3416-3425,doi:10.1096 / fj.11-186346(2011).

[0248] 63 Salker,M.S.et al.Deregulation of the serum-and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure.Naturemedicine 17,1509-1513,doi:10.1038 / nm.2498(2011).

[0249] 64 Kajihara,T.et al.Differential expression of FOXO1 and FOXO3aconfers resistance to oxidative cell death upon endometrialdecidualization.Mol Endocrinol 20,2444-2455,doi:10.1210 / me.2006-0118(2006).

[0250] 65 Muter,J.et al.Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1(PRIP-1)in Decidualizing HumanEndometrial Stromal Cells.Endocrinology 157,2883-2893,doi:10.1210 / en.2015-1914(2016).

[0251] 66 Kuroda,K.et al.Elevated periimplantation uterine natural killercell density in human endometrium is associated with impaired corticosteroidsignaling in decidualizing stromal cells.The Journal of clinicalendocrinology and metabolism 98,4429-4437,doi:10.1210 / jc.2013-1977(2013).

[0252] 67 Weyemi,U.et al.ROS-generating NADPH oxidase NOX4 is a criticalmediator in oncogenic H-Ras-induced DNA damage and subsequentsenescence.Oncogene 31,1117-1129,10 doi:10.1038 / onc.2011.327(2012).

[0253] 68 Colombo,A.R.,Elias,H.K.&Ramsingh,G.Senescence inductionuniversally activates transposable element expression.Cell Cycle,1-12,doi:10.1080 / 15384101.2018.1502576(2018).

[0254] 69 van Deursen,J.M.The role of senescent cells in ageing.Nature 509,439-446,doi:10.1038 / nature13193(2014).

[0255] 70 Uhlen,M.et al.Proteomics.Tissue-based map of the humanproteome.Science 347,1260419,doi:10.1126 / science.1260419(2015).

[0256] 71 van Deursen JM.Senolytic therapies for healthy longevity.Science,364,6441:636-7(2019).

[0257] 72 Barros FS,Brosens,J.J.,Brighton,P.J.Isolation and Primary Cultureof Various Cell Types from Whole Human Endometrial Biopsies.Bio-protocol.2016;6:e2028.

[0258] 73 Masuda H,Anwar SS,Buhring HJ,Rao JR,Gargett CE.A novel marker ofhuman endometrial mesenchymal stem-like cells.Cell Transplant.2012;21(10):2201-14.

[0259] 74 Lucas ES,Dyer NP,Murakami K,Lee YH,Chan YW,Grimaldi G,et al.Lossof Endometrial Plasticity in Recurrent Pregnancy Loss.Stem Cells.2016;34(2):346-56.

[0260] 75 Pfaffl MW.A new mathematical model for relative quantification inreal-time RT-PCR.Nucleic Acids Res.2001;29(9):e45.

[0261] 76 Turco MY,Gardner L,Hughes J,Cindrova-Davies T,Gomez MJ,Farrell L,et al.Long-term,hormone-responsive organoid cultures of human endometrium ina chemically defined medium.Nat Cell Biol.2017.

[0262] 77 Lash GE,Bulmer JN,Li TC,Innes BA,Mariee N,Patel G,etal.Standardisation of uterine natural killer(uNK)cell measurements in theendometrium of women with recurrent reproductive failure.J ReprodImmunol.2016;116:50-9.

[0263] 78 Murakami K,Bhandari H,Lucas ES,Takeda S,Gargett CE,Quenby S,etal.Deficiency in clonogenic endometrial mesenchymal stem cells in obese womenwith reproductive failure--a pilot study.PLoS One.2013;8(12):e82582.

[0264] 79 Wang W,Choi BK,Li W,Lao Z,Lee AY,Souza SC,et al.Quantification ofintact and truncated stromal cell-derived factor-1alpha in circulation byimmunoaffinity enrichment and tandem mass spectrometry.J Am Soc MassSpectrom.2014;25(4):614-25.

[0265] 80 Du H,Taylor HS.Contribution of bone marrow-derived stem cells toendometrium and endometriosis.Stem Cells.2007;25(8):2082-6.

[0266] 81 Morelli SS,Rameshwar P,Goldsmith LT.Experimental evidence for bonemarrow as a source of nonhematopoietic endometrial stromal and epithelialcompartment cells in a murine model.Biol Reprod.2013;89(1):7.

[0267] 82 Taylor HS.Endometrial cells derived from donor stem cells in bonemarrow transplant recipients.JAMA.2004;292(1):81-5.

[0268] 83 Schachinger V,Erbs S,Elsasser A,Haberbosch W,Hambrecht R,Holschermann H,et al.Intracoronary bone marrow-derived progenitor cells inacute myocardial infarction.N Engl J Med.2006;355(12):1210-21.

[0269] 84 Brenner C,Adrion C,Grabmaier U,Theisen D,von Ziegler F,Leber A,etal.Sitagliptin plus granulocyte colony-stimulating factor in patientssuffering from acute myocardial infarction:A double-blind,randomized placebo-controlled trial of efficacy and safety(SITAGRAMI trial).Int J Cardiol.2016;205:23-30.

[0270] 83 Santamaria X,Cabanillas S,Cervello I,Arbona C,Raga F,Ferro J,etal.Autologous cell therapy with CD133+bone marrow-derived stem cells forrefractory Asherman's syndrome and endometrial atrophy:a pilot cohortstudy.Hum Reprod.2016;31(5):1087-96.

[0271] 84 Alba M,Sheng D,Guan Y,Williams-Herman D,Larson P,Sachs JR,etal.Sitagliptin 100mg daily effect on DPP-4inhibition and compound-specificglycemic improvement.Curr Med Res Opin.2009;25(10):2507-14.

[0272] 85 Farag SS,Nelson R,Cairo MS,O'Leary HA,Zhang S,Huntley C,etal.High-dose sitagliptin for systemic inhibition of dipeptidylpeptidase-4toenhance engraftment of single cord umbilical cord bloodtransplantation.Oncotarget.2017;8(66):110350-7.

[0273] Implementation Method

[0274] 1. A method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, thereby assessing the risk, wherein the at least one marker gene for decidual cells comprises phospholipase A2 group IIA (PLA2G2A).

[0275] 2. The method according to embodiment 1, wherein the at least one marker gene of the decidual cells further comprises scavenger receptor class A member 5 (SCARA5), ferritin light chain (FTL), glutaredoxin (GLRX) and / or interleukin 1 receptor-like 1 (IL1RL1).

[0276] 3. The method according to any one of the preceding embodiments, wherein the at least one marker gene of the decidual senescent cells is selected from iodothyronine deiodinase 2 (DIO2), clusterin (CLU), and insulin-like growth factor binding protein 1 (IGFBP1).

[0277] 4. The method according to embodiment 3, wherein the at least one marker gene of decidual senescent cells is DIO2.

[0278] 5. The method according to any one of the preceding embodiments, wherein the method comprises detecting and / or quantifying the amount of PLA2G2A and DIO2.

[0279] 6. The method according to any one of the preceding embodiments, wherein

[0280] Decidual cell marker gene levels are reduced compared to a reference sample or level, and / or

[0281] Increased levels of marker genes for decidual senescent cells compared to a reference sample or level,

[0282] Indicating that the individual is at risk for pregnancy loss or embryo implantation failure.

[0283] 7. The method according to any one of the preceding embodiments, further comprising detecting and / or quantifying the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells in the biological sample.

[0284] 8. The method of embodiment 7, wherein a decreased level of uNK cells or a decreased level of at least one marker gene of uNK cells compared to a reference sample or level indicates that the individual is at risk for pregnancy loss or embryo implantation failure.

[0285] 9. The method according to any one of the preceding embodiments, further comprising the step of determining one or more risk markers selected from the group consisting of maternal body mass index, maternal age, and number of previous pregnancy losses or embryo implantation failures.

[0286] 10. A method for monitoring or evaluating the effectiveness of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effectiveness of the treatment, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

[0287] 11. The method according to embodiment 10, wherein the at least one marker gene of the decidual cells further comprises SCARA5, FTL, GLRX and / or IL1RL1.

[0288] 12. The method according to embodiment 10 or 11, wherein the at least one marker gene of decidual senescent cells is selected from DIO2, CLU and IGFBP1.

[0289] 13. The method of embodiment 12, wherein the at least one marker gene of decidual senescent cells is DIO2.

[0290] 14. The method of any one of embodiments 10 to 13, wherein the method comprises detecting and / or quantifying the amount of PLA2G2A and DIO2.

[0291] 15. The method of any one of embodiments 10 to 14, wherein

[0292] (i) an increase in the level of a marker gene for decidual cells compared to a reference sample or level, and / or a decrease in the level of a marker gene for decidual senescent cells compared to a reference sample or level indicates a positive response to treatment; and

[0293] (ii) decreased levels of marker genes for decidual cells compared to a reference sample or level, and / or increased levels of marker genes for decidual senescent cells compared to a reference sample or level, indicates a negative response to treatment.

[0294] 16. The method of any one of embodiments 10 to 15, further comprising detecting and / or quantifying the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells in the biological sample.

[0295] 17. A method according to embodiment 16, wherein (i) an increase in the level of uNK cells or at least one marker gene of uNK cells compared to a reference sample or level indicates a positive response to treatment; and (ii) a decrease in the level of uNK cells or at least one marker gene of uNK cells compared to a reference sample or level indicates a negative response to treatment.

[0296] 18. The method of any one of embodiments 10 to 17, wherein the method comprises comparing the level of the marker gene at a first time point before or during the treatment with the level of the marker gene at a later time point during or after the treatment.

[0297] 19. The method of any one of the preceding embodiments, wherein the risk of pregnancy loss is the risk of euploid pregnancy loss, or wherein the risk of embryo implantation failure is not caused by a chromosomal abnormality in the embryo.

[0298] 20. The method of any one of the preceding embodiments, wherein the risk of pregnancy loss is the risk of recurrent pregnancy loss.

[0299] 21. A method for diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, thereby diagnosing the disorder, wherein the at least one marker for decidual cells comprises PLA2G2A.

[0300] 22. The method of embodiment 21, wherein the reproductive disorder is embryo implantation failure, pregnancy loss, recurrent miscarriage, recurrent pregnancy loss, or placental disease.

[0301] 23. The method of embodiment 21 or embodiment 22, wherein the at least one marker gene of the decidual cells further comprises SCARA5, FTL, GLRX and / or IL1RL1.

[0302] 24. The method of any one of embodiments 21 to 23, wherein the at least one marker gene of decidual senescent cells is selected from the group consisting of DIO2, CLU, and IGFBP1.

[0303] 25. The method of embodiment 24, wherein the at least one marker gene for decidual senescent cells is DIO2.

[0304] 26. The method of any one of embodiments 21 to 25, wherein the method comprises detecting and / or quantifying the amount of PLA2G2A and DIO2.

[0305] 27. The method of any one of embodiments 21 to 26, wherein

[0306] The level of the decidual cell marker gene is reduced compared to a reference sample or level, and / or

[0307] The level of the decidual senescent cell marker gene is increased compared to a reference sample or level,

[0308] Indicates a positive diagnosis.

[0309] 28. The method of any one of embodiments 21 to 27, further comprising detecting and / or quantifying the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells in the biological sample.

[0310] 29. The method of embodiment 28, wherein a decrease in the level of uNK cells or at least one marker gene of uNK cells compared to a reference sample or level indicates a positive diagnosis.

[0311] 30. The method of any one of embodiments 27 to 29, wherein in a positive diagnosis the ratio of PLA2G2A to DIO2 is:

[0312] (i) below the 50th percentile compared to a reference sample or reference level; or

[0313] (ii) below the 30th percentile compared to a reference sample or reference level.

[0314] 31. The method of any one of the preceding embodiments, wherein the biological sample is an endometrial biopsy sample.

[0315] 32. The method of any one of the preceding embodiments, wherein the biological sample is collected during the luteal phase of the menstrual cycle, optionally wherein the biological sample is collected during the mid-luteal phase of the menstrual cycle.

[0316] 33. The method of any one of the preceding embodiments, wherein the individual suffers from or has suffered from infertility or embryo implantation failure following in vitro fertilization treatment.

[0317] 34. The method of any one of the preceding embodiments, wherein the individual has suffered at least one previous pregnancy loss or embryo implantation failure, or has suffered recurrent pregnancy loss.

[0318] 35. The method of any one of the preceding embodiments, wherein the method further comprises detecting and / or quantifying genes that allow identification of days in the menstrual cycle, optionally wherein the genes that allow identification of the timing of days in the menstrual cycle comprise, consist of or consist essentially of glutathione peroxidase 3 (GPX3) and solute carrier family 15 member 2 (SLC15A2).

[0319] 36. The method of any one of the preceding embodiments, wherein the marker genes are detected and / or quantified using ELISA, Western blot, immunohistochemistry, immunoassay, enzyme assay, or sequencing, optionally wherein the sequencing method comprises qPCR, Taqman-PCR, multiplex Taqman-PCR, Nanostring, targeted sequencing, or digital PCR.

[0320] 37. A method according to embodiment 36, wherein the digital PCR is digital droplet PCR (ddPCR).

[0321] 38. A method of treating a reproductive disorder in an individual, the method comprising diagnosing the reproductive disorder according to any one of the methods of embodiments 21 to 37, and administering an agent or implementing a treatment regimen to effectively treat the reproductive disorder in the individual diagnosed positive.

[0322] 39. The method of embodiment 38, wherein the agent or treatment regimen increases the level of decidual cells and / or uNK cells in the individual, and / or decreases the level of decidual senescent cells in the individual.

[0323] 40. The method of embodiment 38 or 39, wherein the agent is a DPP4 inhibitor.

[0324] 41. The method of embodiment 40, wherein the DPP4 inhibitor is sitagliptin.

[0325] 42. The method of any one of embodiments 38 to 41, wherein the individual has an increased level of at least one marker gene for decidual senescent cells.

[0326] 43. The method of any one of embodiments 38 to 42, wherein the at least one marker gene for decidual senescent cells is DIO2.

[0327] 44. The method according to any one of embodiments 38 to 43, comprising administering a progestin and / or a progestogen.

[0328] 45. The method according to any one of embodiments 21 to 44, wherein the reproductive disorder is recurrent pregnancy loss.

[0329] 46. A method for selecting a patient for treatment to reduce the risk of embryonic implantation failure or pregnancy loss, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from an individual, and selecting the patient for treatment to reduce the risk of pregnancy loss or embryonic implantation failure based on the levels of the marker genes, wherein the at least one marker for decidual cells comprises PLA2G2A.

[0330] 47. The method of embodiment 46, wherein an individual in whom an increased level of at least one marker gene for decidual senescent cells is detected is selected to receive treatment.

[0331] 48. The method of embodiment 47, wherein the patient is selected to receive treatment with a DPP4 inhibitor.

[0332] 49. The method of embodiment 48, wherein the DPP4 inhibitor is sitagliptin.

[0333] 50. The method of any one of embodiments 46 to 49, wherein the at least one marker gene for decidual senescent cells is DIO2.

[0334] 51. The method of embodiments 46 to 50, wherein the method of selecting a patient for treatment is to reduce the risk of pregnancy loss, and wherein the pregnancy loss is recurrent pregnancy loss.

[0335] 52. A detection kit suitable for use in the method of any one of the preceding embodiments, wherein the detection kit comprises a device for detecting or quantifying at least one marker gene of decidual cells and at least one marker gene of decidual senescent cells at the nucleic acid or protein level, and optionally a device for detecting and / or quantifying the level of uNK cells in the individual or the level of at least one marker gene of uNK cells, wherein the at least one marker gene of decidual cells comprises PLA2G2A.

Claims

1. A method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, thereby assessing the risk, wherein the at least one marker gene for decidual cells comprises phospholipase A2 group IIA (PLA2G2A).

2. The method according to claim 1, wherein (a) the at least one marker gene of the decidual cells further comprises scavenger receptor class A member 5 (SCARA5), ferritin light chain (FTL), glutaredoxin (GLRX) and / or interleukin 1 receptor-like 1 (IL1RL1); and / or (b) the at least one marker gene of the decidual senescent cells is selected from iodothyronine deiodinase 2 (DIO2), clusterin (CLU) and insulin-like growth factor binding protein 1 (IGFBP1), optionally wherein the at least one marker gene of the decidual senescent cells is DIO2; and / or (c) The method comprises detecting and / or quantifying the amount of PLA2G2A and DIO2.

3. A method according to any one of the preceding claims, wherein The level of a marker gene in the decidual cells is reduced compared to a reference sample or level, and / or The level of a marker gene for decidual senescent cells is increased compared to a reference sample or level, Indicating that the individual is at risk for pregnancy loss or embryo implantation failure.

4. The method according to any one of the preceding claims, further comprising: (a) detecting and / or quantifying the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells in the biological sample, optionally wherein a decreased level of uNK cells or the level of at least one marker gene for uNK cells compared to a reference sample or level indicates that the individual is at risk of pregnancy loss or embryo implantation failure; and / or (b) determining one or more risk markers selected from the group consisting of maternal body mass index, maternal age, and number of previous pregnancy losses or embryo implantation failures.

5. A method for monitoring or evaluating the effectiveness of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effectiveness of the treatment, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

6. The method according to claim 5, wherein (a) the at least one marker gene of the decidual cells further comprises SCARA5, FTL, GLRX and / or IL1RL1; and / or (b) the at least one marker gene of the decidual senescent cells is selected from DIO2, CLU and IGFBP1, optionally wherein the at least one marker gene of the decidual senescent cells is DIO2; and / or (c) The method comprises detecting and / or quantifying the amount of PLA2G2A and DIO2.

7. The method according to claim 5 or 6, wherein (i) an increase in the level of a marker gene for decidual cells compared to a reference sample or level, and / or a decrease in the level of a marker gene for decidual senescent cells compared to a reference sample or level, indicating a positive response to treatment; and (ii) a decrease in the level of a marker gene for decidual cells compared to a reference sample or level, and / or an increase in the level of a marker gene for decidual senescent cells compared to a reference sample or level, indicating a negative response to treatment.

8. The method according to any one of claims 5 to 7, wherein (a) the method further comprises detecting and / or quantifying the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells in the biological sample, optionally wherein (i) an increase in the level of uNK cells or the at least one marker gene for uNK cells, compared to a reference sample or level, indicates a positive response to the treatment; and (ii) a decrease in the level of uNK cells or the at least one marker gene for uNK cells, compared to a reference sample or level, indicates a negative response to the treatment; and / or (b) The method comprises comparing the level of the marker gene at a first time point before or during the treatment with the level of the marker gene at a later time point during or after the treatment.

9. A method according to any one of the preceding claims, wherein (a) the risk of pregnancy loss is the risk of euploid pregnancy loss, or wherein the risk of embryonic implantation failure is not due to a chromosomal abnormality in the embryo; and / or (b) the risk of pregnancy loss is the risk of recurrent pregnancy loss.

10. A method for diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from the individual, thereby diagnosing the disorder, wherein the at least one marker for decidual cells comprises PLA2G2A.

11. The method according to claim 10, wherein (a) the reproductive disorder is implantation failure, pregnancy loss, recurrent miscarriage, recurrent pregnancy loss or placental disease; and / or (b) the at least one marker gene of the decidual cells further comprises SCARA5, FTL, GLRX and / or IL1RL1; and / or (c) the at least one marker gene of the decidual senescent cells is selected from DIO2, CLU and IGFBP1, optionally wherein the at least one marker gene of the decidual senescent cells is DIO2; and / or (d) The method comprises detecting and / or quantifying the amount of PLA2G2A and DIO2.

12. The method according to claim 10 or 11, wherein The level of the decidual cell marker gene is reduced compared to a reference sample or level, and / or The level of the decidual senescent cell marker gene is increased compared to a reference sample or level, Indicates a positive diagnosis.

13. The method according to any one of claims 10 to 12, further comprising detecting and / or quantifying the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells in the biological sample, optionally wherein a reduced level of uNK cells or at least one marker gene for uNK cells compared to a reference sample or level indicates a positive diagnosis.

14. The method according to claim 12 or 13, wherein in a positive diagnosis the ratio of PLA2G2A to DIO2 is: (i) below the 50th percentile compared to a reference sample or reference level; or (ii) below the 30th percentile compared to a reference sample or reference level.

15. A method according to any one of the preceding claims, wherein (a) the biological sample is an endometrial biopsy sample; and / or (b) the biological sample is collected during the luteal phase of the menstrual cycle, optionally wherein the biological sample is collected during the mid-luteal phase of the menstrual cycle.

16. A method according to any one of the preceding claims, wherein the individual (a) suffers or has suffered from infertility or embryo implantation failure after in vitro fertilization treatment; and / or (b) have had at least one previous pregnancy loss or embryo implantation failure, or have had recurrent pregnancy loss.

17. A method according to any one of the preceding claims, wherein (a) the method further comprises detecting and / or quantifying a gene that allows identification of the day in the menstrual cycle, optionally wherein the gene that allows identification of the timing of the day in the menstrual cycle comprises, consists of or consists essentially of glutathione peroxidase 3 (GPX3) and solute carrier family 15 member 2 (SLC15A2); and / or (b) detecting and / or quantifying the marker genes using ELISA, western blot, immunohistochemistry, immunoassay, enzyme assay or sequencing method, optionally wherein the sequencing method comprises qPCR, Taqman-PCR, multiplex Taqman-PCR, Nanostring, targeted sequencing or digital PCR, optionally wherein the digital PCR is digital droplet PCR (ddPCR).

18. A method of treating a reproductive disorder in an individual, the method comprising diagnosing the reproductive disorder according to any one of claims 10 to 17, and administering a pharmaceutical agent or implementing a treatment regimen to effectively treat the reproductive disorder in the individual diagnosed positive.

19. The method according to claim 18, wherein (a) the agent or treatment regimen increases the level of decidual cells and / or uNK cells in the individual, and / or decreases the level of decidual senescent cells in the individual; (b) the agent is a DPP4 inhibitor, optionally wherein the DPP4 inhibitor is sitagliptin; and / or (c) the individual has an increased level of at least one marker gene for decidual senescent cells; and / or (d) the at least one marker gene of the decidual senescent cells is DIO2; and / or (e) The method comprises administering progesterone and / or progestogen.

20. The method of any one of claims 10-19, wherein the reproductive disorder is recurrent pregnancy loss.

21. A method for selecting a patient for treatment to reduce the risk of embryonic implantation failure or pregnancy loss, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells in a biological sample obtained from an individual, and selecting the patient for treatment to reduce the risk of pregnancy loss or embryonic implantation failure based on the levels of the marker genes, wherein the at least one marker for decidual cells comprises PLA2G2A.

22. The method of claim 21, wherein an individual in whom increased levels of at least one marker gene for decidual senescent cells are detected is selected to receive treatment, optionally wherein the patient is selected to receive treatment with a DPP4 inhibitor, further optionally wherein the DPP4 inhibitor is sitagliptin.

23. The method according to claim 21 or 22, wherein (a) at least one marker gene of the decidual senescent cells is DIO2; and / or (b) The method of selecting a patient for treatment is to reduce the risk of pregnancy loss, and wherein the pregnancy loss is recurrent pregnancy loss.

24. A detection kit suitable for use in the method of any of the preceding claims, wherein the detection kit comprises a device for detecting or quantifying at the nucleic acid or protein level at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells, and optionally a device for detecting and / or quantifying the level of uNK cells or the level of at least one marker gene for uNK cells in the individual, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

Images