Biomarkers of pregnancy loss

Abstract

The present invention also relates to methods for assessing the risk of pregnancy loss or embryo implantation failure, and to methods for monitoring or evaluating the effectiveness of treatments to reduce the risk of pregnancy loss or embryo implantation failure using specific biomarkers. The present invention also relates to the use of these biomarkers in methods for diagnosing reproductive disorders in individuals, as well as methods for treating reproductive disorders. Furthermore, the biomarkers can further 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.

Description

[Technical Field]

[0001] The present invention relates to methods for assessing the risk of pregnancy loss or embryo implantation failure, and to methods for monitoring or evaluating the effectiveness of treatments to reduce the risk of pregnancy loss or embryo implantation failure using specific biomarkers. The present invention also relates to the use of these biomarkers in methods for diagnosing reproductive disorders in individuals, as well as methods for treating reproductive disorders. Furthermore, the biomarkers can further 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 technology]

[0002] Approximately 15% of clinical pregnancies end in pregnancy loss, usually during the first trimester. 1 Fetal chromosomal abnormalities account for 50-60% of miscarriages. 2、3 The incidence of aneuploid pregnancies is increasing in developed countries, paralleling a demographic shift toward older maternal age. 2 Aneuploid pregnancy loss is defined as two or more losses. 9、10 Less common in recurrent pregnancy loss (RPL) 4~8 With each pregnancy loss, the frequency of euploid loss increases, but the chances of a successful pregnancy decrease. 4 These findings indicate that maternal factors contribute to high pregnancy loss, but few interventions have been shown to improve live birth rates in RPL. 9 , often reflecting that the underlying mechanisms are unknown.

[0003] Increasing evidence from animal studies suggests that dysregulated interactions between the conceptus and endometrium at the time of implantation cause a ripple effect that ultimately leads to pregnancy failure 11~13 Implantation requires intense remodeling of the endometrial stroma, triggered by the postovulatory surge in progesterone and elevated intracellular cyclic adenosine monophosphate levels. 14This process, termed decidualization, is initiated during the mid-luteal phase of each cycle and involves the differentiation and polarization of endometrial stromal cells (EnSCs) into stress-resistant and stressed / senescent decidual subpopulations. 15 Concurrently, uterine natural killer (uNK) cells accumulate in the stroma and, in response to IL-15 activation, target and eliminate stressed decidual cells. During implantation, embryo-derived chorionic gonadotropin rescues the corpus luteum, and sustained progesterone signaling promotes the formation of a tightly bound, immune-privileged decidual matrix around the conceptus. 16 Therefore, a key challenge during implantation is to simultaneously avoid impending endometrial breakdown while converting the cyclic endometrium into a semi-permanent tissue, namely the 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). Briefly, balancing decidual subpopulations at implantation each cycle is controlled by the influx of MSCs, allowing for the expansion of the pool of uterine NK cells that target and eliminate decidual cells and stressed / senescent cells in early pregnancy. Recurrent pregnancy loss is associated with a deficiency of both MSCs and uNK cells. 15、18~19 (Brighton et al., 2017, Lucas et al., 2016, Lucas et al., 2020). Importantly, the level of depletion correlates with the number of previous miscarriages and therefore the risk of recurrence. 18、20 (Lucas et al., 2016, Tewary et al., 2020).

[0005] Preconception screening and intervention can reduce the suffering of pregnancy loss. However, currently, there is no diagnostic test to (i) assess the role of endometrial dysfunction in recurrent pregnancy loss, (ii) evaluate the effectiveness or therapeutic intervention before conception, and (iii) inform patients and their physicians of their risk for future loss of a chromosomally normal pregnancy. The Implantation Clinic at the Biomedical Research Unit in Reproductive Health (UHCW) currently offers a test based on 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, the test is performed on biopsies obtained over two menstrual cycles. Patients are either self-referred or referred by their general practitioner. The diagnostic pathway is complex, labor-intensive, and dependent on skilled staff. Therefore, test results are only available 5–6 weeks later. Another significant drawback of current uNK cell tests is their reliance on indirect measurements of endometrial "status."

[0006] However, a major challenge in the clinical management of recurrent pregnancy loss is distinguishing between embryonic and maternal causes of pregnancy failure. Two independent risk factors, maternal age and the number of previous pregnancy losses, have unbalanced effects on pregnancy loss rates (Magnus et al., 2019). The age-specific risk of pregnancy loss is explained by the increased incidence of meiotic errors in oocytes, which leads to a rapid increase in aneuploid embryos after the age of 35. Meanwhile, the risk of recurrent pregnancy loss increases stepwise by approximately 10% with each additional pregnancy loss, regardless of maternal age (Magnus et al., 2019). Therefore, an essential criterion for any clinical trial aimed at identifying maternal factors causally related to pregnancy loss is that the frequency of positive test results must increase with each additional pregnancy loss, regardless of maternal age. None of the current pregnancy loss tests in clinical practice have been shown to meet this basic criterion for causality.

[0007] The lack of decidual cells and the excess of stressed / senescent stromal cells result in an embryo with an endometrial environment that is vulnerable, lacks biosensing properties, and is prone to collapse. At a functional level, this pathological endometrial state has been termed "implantation checkpoint failure," which means that the endometrium is unable to select for low-fitness embryos or adequately support high-quality embryos. Both scenarios 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 of specific endometrial stromal and epithelial subpopulations during the peri-implantation window. We previously discovered (as described in WO 2021 / 032973) that abnormal levels of markers of decidual cells and / or senescent decidual cells, particularly SCARA5 and DIO2, are associated with reproductive defects such as miscarriage. Summary of the Invention

[0009] The discovery of specific biomarkers for decidual cells and decidual senescent cells allows for the assessment of the "state" of the endometrium during the mid-luteal implantation window before conception and makes it possible to identify women at risk of subsequent pregnancy loss caused by impaired endometrial function. The inventors surprisingly identified PLA2G2A as a biomarker for pregnancy loss, particularly recurrent pregnancy loss. The biomarkers of the present invention are also advantageous compared to previously described biomarkers, such as SCARA5, because the greater dynamic range of PLA2G2A expression (as illustrated in Figure 2B) enhances sensitivity. The development of clinical trials based on the biomarkers described herein, including the detection of PLA2G2A, can be used in the management of women with a history of one or more previous pregnancy losses. This test can also be used to evaluate the effectiveness of preconception interventions, such as drug treatments, aimed at reducing the risk of pregnancy loss and increasing live birth rates.

[0010] The test of the present invention is specifically designed to evaluate the endometrial causes of pregnancy loss before conception and assist / instruct therapeutic intervention.Due to its simplicity and ease, it can become a routine, quick and relatively inexpensive test for any woman who suffers from pregnancy loss, even if it is the first time.This is an easier and cheaper alternative to the currently available commercial test (described in EP2333107B1) that aims to determine the "implantation window" in IVF patients.Importantly, the test of the present invention shows an increased frequency of positive results with each additional pregnancy loss, regardless of maternal age, and therefore can be said to identify the maternal factors that are causally related to miscarriage.

[0011] Therefore, the present inventors have surprisingly found that PLA2G2A can be used as a biomarker, either alone or in combination, in the method for assessing the risk of pregnancy loss or embryo implantation failure, and also in the method for monitoring or evaluating the effect of treatment for reducing the risk of pregnancy loss or embryo implantation failure.The biomarkers described herein can also be used in the method for diagnosing reproductive disorders in individuals, and also to assist in the method for treating reproductive disorders.In addition, these biomarkers can also be used in the method for selecting patients for treatment for reducing the risk of embryo implantation failure or miscarriage, or in the method for stratifying patients.Biomarkers can be detected using the kit described herein.

[0012] The present invention therefore provides a method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, comprising the step of 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 PLA2G2A.

[0013] The present invention also provides a method for monitoring or assessing the effectiveness of a treatment for reducing the risk of pregnancy loss or embryo implantation failure in an individual, the method comprising the step of 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 monitoring or assessing the effectiveness of the treatment, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

[0014] The present invention further provides a method for diagnosing a reproductive disorder in an individual, the method 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 the individual, thereby diagnosing the disorder, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

[0015] The present invention also provides methods for treating a reproductive disorder in an individual or for preventing pregnancy loss or embryonic implantation failure in an individual, comprising the steps of diagnosing the reproductive disorder or assessing the risk of pregnancy loss or embryonic implantation failure as described herein, and administering an agent or treatment regimen effective to treat the reproductive disorder or prevent pregnancy loss or embryonic implantation failure in an individual positively diagnosed 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, 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, 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 for decidual cells comprises PLA2G2A.

[0017] The present invention also provides a test kit suitable for use in the methods described herein, comprising means 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 means for detecting and / or quantifying in an individual the level of uNK cells or the level of at least one marker gene for uNK cells.

[0018] The present invention further provides a method for assessing readiness for conception or successful embryo implantation in an individual, the method 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 the individual, thereby assessing readiness for conception or successful embryo implantation, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

[0019] The present invention provides an endometrial test that is particularly advantageous for patients with recurrent pregnancy loss. The test is suitable for any woman who is planning to become pregnant. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 illustrates mechanisms affecting the decidual pathway. The transition of cyclic endometrium to decidualization during pregnancy requires the cooperation of decidual cells and uterine natural killer (uNK) cells to eliminate senescent decidual cells. Concurrently, bone marrow-derived mesenchymal stem cells (MSCs) are recruited to ensure rapid tissue expansion during pregnancy. The lack of MSCs and / or uNK cells renders the uteroplacental interface susceptible to tissue breakdown during pregnancy, thereby driving a pro-senescence decidual response that leads to miscarriage. SASP, senescence-associated secretory phenotype; IL-15, interleukin-15. [Figure 2-1]Figure 2 shows the expression of decidual cell markers and decidual senescent cell markers in the endometrium. (A) SCARA5 and DIO2 percentiles for six paired endometrial biopsies (n = 12) subjected to RNA-seq. Open and closed circles indicate "A" and "B" biopsies, respectively. Paired biopsies are indicated by dashed lines. Samples in the upper left quadrant (SCARA5 mRNA levels < 30th percentile and DIO2 > 70th percentile) were considered "abnormal," i.e., possessing excessive stressed / senescent cells and lacking decidual cells. (B) A volcano plot showing differential gene expression between endometrial samples considered "normal" and "abnormal" reveals that high DIO2 expression is associated with low PLA2G2A expression, and vice versa. A Bonferroni correction at 0.05 is indicated by the dashed line. (C) Violin plot showing mRNA expression of DIO2 and PLA2G2A in endothelial, epithelial, immune, and stromal cells from single cells in vivo in luteal phase endometrial biopsies. [Figure 2-2] Figure 2 shows the expression of decidual cell markers and decidual senescent cell markers in the endometrium (continued). (D) mRNA expression of DIO2 and PLA2G2A in the endometrium during the proliferative phase and the early, mid, and late luteal phases. Each bar represents an individual biopsy. Data were obtained from microarray data deposited in Gene Expression Omnibus (GEO profile ID: GDS2052). (E) mRNA levels of DIO2 and PLA2G2A were quantified by RT-qPCR analysis in 822 endometrial biopsies obtained between LH+6 and LH+11. Percentile graphs showing the distribution of gene expression across the peri-implantation window were generated based on normalized expression values ​​using R software. The median number of samples on each day was 153 (range: 51-202). [Figure 3]Figure 3 shows the spatial organization of PLA2G2A- and DIO2-expressing cells in the endometrium. Spot plots showing log-transformed, normalized spatial expression of DIO2 (top panel) and PLA2G2A (bottom panel) in formalin-fixed, paraffin-embedded endometrial tissue sections (LH+8), original magnification 10x. [Figure 4] Figure 4 shows the association between the PLA2G2A / DIO2 ratio and the number of previous miscarriages. (A) Distribution of PLA2G2A / DIO2 percentiles in 854 LH-timed endometrial biopsies from patients with a history of 0 to 18 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 the number of previous miscarriages. (C) Frequency of endometrial samples with a PLA2G2A / DIO2 ratio <15th percentile as a function of the number of previous losses. Total n numbers are shown for each column. 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 day of biopsy after luteinizing hormone surge (LH+, lower panel) between samples with PLA2G2A / DIO2 ratios >15th percentile (designated "normal") and <15th centile ("abnormal"). Student's t-test; ** indicates P<0.01; ns: not significant (P>0.05). [Figure 5]Figure 5 shows the use of the PLA2G2A / DIO2 ratio to predict risk of future pregnancy loss. (A) Analysis of the PLA2G2A / DIO2 ratio in endometrial biopsies obtained before pregnancies resulting in live birth or miscarriage. The left panel shows the relative rates of live births vs. pregnancy losses in prepregnancy endometrial biopsies with PLA2G2A / DIO2 ratios below the indicated percentile. The right panel shows the relative rates of live births vs. pregnancy losses in prepregnancy endometrial biopsies with PLA2G2A / DIO2 ratios above the indicated percentile. 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 the subsequent pregnancy resulting in live birth or miscarriage. Student's t-test; ns: not significant (P>0.05). [Figure 6] Figure 6 shows the effect of oral sitagliptin on the endometrial PLA2G2A / DIO2 ratio. DIO2 and PLA2G2A transcripts were measured by RT-qPCR in paired baseline and second endometrial biopsies obtained from participants in the placebo (n = 16) and sitagliptin (n = 15) groups of the SIMPLANT trial (EudraCT number 2016-001120-54). The PLA2G2A / DIO2 ratio (lower panel) was calculated. Data were analyzed using a Wilcoxon matched-pairs signed-rank test with Sidak correction. DETAILED DESCRIPTION OF THE INVENTION

[0021] It is understood that various applications of the disclosed methods can be tailored to specific needs in the art. It is also understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. All publications, patents, and patent applications cited herein, whether preceded or followed, are hereby incorporated by reference in their entirety.

[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes "cells," etc.

[0023] Methods for assessing risk of pregnancy loss or embryo implantation The present invention provides a method for assessing the risk (or possibility or probability) of pregnancy loss or embryo implantation failure in an individual, 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 the individual, thereby assessing the risk, wherein the at least one marker for 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, at least one of the decidual marker genes detected is PLA2G2A. Therefore, the decidual marker gene used for detection comprises at least PLA2G2A. The method can further comprise detecting additional decidual marker genes.

[0024] Any additional marker gene for decidual cells and at least one marker gene for decidual senescent cells can be selected from any such marker gene. Thus, the marker gene can be any gene that indicates the level of decidual cells and decidual senescent cells in a sample, such as an endometrial sample. For example, the marker gene for decidual cells can be any marker whose decrease indicates a decreased level of decidual cells, and the marker gene for decidual senescent cells can be any marker whose increase indicates an increased 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.

[0025] The level of the marker gene is typically compared with a control or reference sample or level. Any suitable control or reference sample or level can be used. The control or reference sample or level can refer to, for example, a normal or healthy sample / level obtained or determined from one or more individuals who do not have any reproductive disorder or have never experienced pregnancy loss or embryo implantation failure. The individual may have experienced one or more successful pregnancies. The control or reference sample or level can also be obtained or determined from one or more individuals who have responded positively to a treatment to reduce the risk of pregnancy loss or embryo implantation failure. Alternatively, the control or reference sample or level can refer to a sample / level from one or more individuals who have a reproductive disorder or have experienced one or more miscarriages or embryo implantation failure, i.e., a positive control or reference sample or level. In the case of control or reference samples or levels from multiple individuals, an average value can be obtained, or the results can be pooled to generate a more accurate reference range. The level determined in the test sample is preferably compared to a control or reference sample or level obtained on or about the same day of the menstrual cycle as the test sample. Other suitable control or reference samples or levels can be readily identified by one of skill in the art.

[0026] Compared with a reference sample or level, a decreased or decreased level of a marker gene for decidual cells, such as PLA2G2A, in a sample can indicate that the individual is at risk of pregnancy loss or embryo implantation failure. Alternatively, compared with a reference sample or level, an increased or increased level of a marker gene for decidual senescent cells, such as DIO2, can indicate that the individual is at risk of pregnancy loss or embryo implantation failure. The level of a marker gene can be determined using any method described herein and known to those skilled in the art. For example, compared with a reference sample or level, a decreased or decreased level of a marker gene for decidual cells, such as PLA2G2A, and compared with a reference sample or level, an increased or increased level of a marker gene for decidual senescent cells, such as DIO2, both indicate that the individual is at risk of pregnancy loss or embryo implantation failure.

[0027] As an example, a method for assessing the risk of pregnancy loss or embryonic implantation failure in an individual further includes detecting and / or quantifying the level of uterine natural killer (uNK) cells in the sample, e.g., based on the level of at least one marker gene for uNK cells in the sample.

[0028] A decreased or decreased level of uNK cells or uNK cell gene markers in a sample compared to a reference sample or level can indicate that the individual is at risk for pregnancy loss or embryonic implantation failure.

[0029] The method of the present invention may further comprise the step of detecting and / or quantifying genes that allow identification of the days in the menstrual cycle as described below.

[0030] Additional risk indicia that can be used to assess the risk of pregnancy loss or embryonic implantation failure include maternal body mass index (BMI), maternal age, number of previous pregnancy losses or embryonic implantation failures, familial and intergenerational factors, history of infertility, placental abnormalities, cervical and uterine abnormalities, smoking, alcohol use, etc. Additional risk indicia are known to those skilled in the art.

[0031] For example, there may be an increased risk of pregnancy loss or embryo implantation failure if the maternal BMI is too low (e.g., <18.5) or too high (e.g., >25), if the mother is 35 years of age or older, or if there is a history of recurrent pregnancy loss (e.g., if the woman has had two or more pregnancy losses before reaching 20 weeks of pregnancy).

[0032] sample As used herein, the term "biological sample" or "sample" refers to any sample obtained from an individual. Suitable samples for the methods of the present invention include, for example, endometrial tissue, endometrial secretions, cells obtained from the endometrium, or endometrial biopsy samples.

[0033] Samples obtained from the endometrium can be collected by any method known in the art, including through endometrial biopsy or endometrial sampling. The technique involves removing a portion of tissue from the inner lining of the uterus (endometrium). Samples can be obtained using a uterine evacuation procedure.

[0034] The sample may be or have been processed prior to use, for example by dilution, centrifugation, or extraction of DNA, RNA, or protein. The sample may be a freshly obtained sample or may have been stored or preserved, for example by freezing, prior to use.

[0035] The sample 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 in other phases of the menstrual cycle. The sample can be collected during the mid-luteal phase of the menstrual cycle. Therefore, the sample is typically collected after ovulation. The sample is typically collected during the embryo implantation window (also known as the receptive window) of the menstrual cycle, during which the endometrium is receptive to embryo implantation. The embryo implantation window can be determined by any means, for example, can be calculated based on an ovulation test. The ovulation test can be based on hormone levels, such as luteinizing hormone (LH) levels (e.g., LH levels in urine) or estrogen levels (e.g., based on salivary ferning). Alternatively, the level of one or more markers indicating receptivity to embryo implantation can be determined. Samples can be taken from about 5 to about 11 days after an increase or surge in LH levels (e.g., a 2- to 5-fold increase or surge), i.e., from LH+5 to LH+11. LH is produced by the pituitary gland and is generally secreted at very low levels throughout the menstrual cycle, however, the ovulatory phase of the menstrual cycle begins with an LH surge.

[0036] The above sample types and timing of sample collection are applicable to any of the methods of the invention for detecting marker levels.

[0037] individual The individual referred to in any of the methods of the present invention may be a human or a menstruating non-human mammal. Thus, the methods described herein can be applied in veterinary settings. The subject is preferably a human female.

[0038] The individual may suffer from or have suffered from infertility or embryo implantation failure.For example, the individual may suffer from or have suffered from embryo implantation failure after IVF treatment.The individual may have suffered from at least one previous pregnancy loss or multiple pregnancy losses, and / or at least one previous embryo implantation failure or multiple embryo implantation failures.The individual may suffer from recurrent pregnancy loss (RPL).

[0039] Individual may be considered to be at risk of pregnancy loss or embryo implantation failure.Individual may be considered to be at risk of pregnancy loss or embryo implantation failure because of the existence of one or more risk symptoms, such as low or high body mass index (BMI), maternal age, the number of previous pregnancy loss or embryo implantation failure, familial and intergenerational factors, infertility history, placental abnormality, cervical and uterine abnormality, etc.Other risk symptoms of pregnancy loss or embryo implantation failure are known to those skilled in the art.

[0040] pregnancy loss Pregnancy loss refers to the possibility that the embryo implantation was unsuccessful and the embryo does not result in an infant. Pregnancy loss assessed by the biomarkers of the present invention is typically loss in the first trimester, particularly in the first 20-23 weeks of gestation, which may also be referred to as "miscarriage." Typical symptoms of miscarriage include vaginal bleeding with or without pain, as well as lower abdominal cramps and pain. If an individual has had at least two miscarriages, they may be diagnosed as suffering from recurrent pregnancy loss (RPL) or infertility. However, RPL may not be a binary condition, and any previous loss may increase the risk of further pregnancy loss. The present invention enables the detection of any predisposition to RPL, regardless of the number of previous losses.

[0041] The majority of miscarriages are thought to be caused by chromosomal abnormalities or errors in the embryo, such as aneuploidy, e.g., autosomal trisomy, monosomy X, triploidy, tetraploidy, etc. Other miscarriages are not due to chromosomal abnormalities or errors in the embryo.

[0042] The method of the present invention can assess the risk of pregnancy loss / miscarriage.The method of the present invention is preferably used to determine the risk of euploid miscarriage, and is used when the cause of pregnancy loss is not due to chromosomal abnormality or error in embryo.The method of the present invention is preferably used to determine the risk of recurrent pregnancy loss / miscarriage.

[0043] Embryo implantation failure After fertilization, the fertilized egg (or zygote) begins to divide by mitosis to produce an embryo. The process by which the embryo attaches to the lining of the uterus, i.e., the endometrium, is known as implantation. "Embryonic implantation failure" or "implantation failure" according to the present invention refers to the failure of the embryo to implant in the endometrium. Implantation failure can occur when a patient is trying to conceive naturally without any infertility treatment, or after undergoing assisted reproductive technology, such as in vitro fertilization (IVF).

[0044] Some cases of implantation failure are thought to be caused by chromosomal abnormalities in the embryo, such as aneuploidy, such as autosomal trisomy, monosomy X, triploidy, tetraploidy, etc. Other cases of embryo implantation failure are not caused by chromosomal abnormalities or errors in the embryo. Some cases of IVF-related implantation failure are caused by poor embryo quality, egg age, lack of response to IVF medication, or other lifestyle factors (e.g., smoking).

[0045] According to the present invention, the risk of any case of embryo implantation failure can be evaluated in any situation.Therefore, the method of the present invention for evaluating the risk of embryo implantation failure can be used to determine the risk of implantation failure in an individual after natural conception or after assisted reproduction, such as in vitro fertilization.Preferably, this method is used to determine the risk of embryo implantation failure that is not caused by chromosomal abnormalities or errors in the embryo.

[0046] Decidual cells and decidual senescent cells and marker genes According to the method of the present invention for assessing the risk of pregnancy loss or implantation failure, at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells are detected and / or quantified, and the at least one marker gene for decidual cells includes PLA2G2A.

[0047] Upon exiting the cell cycle, cells either differentiate into specialized cells or undergo senescence. Cellular senescence is a defined cellular state that can be acute or chronic. A key feature of senescent cells (SNC) is 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," termed the senescence-associated secretory phenotype (SASP), which can disrupt normal tissue architecture and function through diverse mechanisms, including recruitment of inflammatory immune cells, extracellular matrix remodeling, induction of fibrosis, and inhibition of stem cell function. 58 .

[0048] During decidualization, endometrial cells undergo significant changes in preparation for and during pregnancy. During the process, endometrial stromal cells (EnSCs) either become specialized cells (i.e., decidual cells) or undergo acute senescence (i.e., decidual SNCS).

[0049] The detected decidual cells may be any decidual cells, and the detected decidual senescent cells may be any decidual senescent cells, typically any such cells in an endometrial sample. Decidua or decidual senescent cells are typically derived from endometrial stromal cells. Decidual cells are stress-resistant and are also referred to herein as stress-resistant decidual cells.

[0050] According to the method of the present invention for assessing the risk of pregnancy loss or implantation failure, at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells can be detected, and the at least one marker gene for decidual cells includes PLA2G2A. The at least one marker gene for decidual cells can further include one or more of SCARA5, FTL, GLRX, and IL1RL1, and the marker gene for decidual senescent cells typically includes one or more of DIO2, CLU, and IGFBP1. The method can include detecting a decrease in PLA2G2A. Preferably, the at least one marker gene for decidual senescent cells is DIO2. The method can include detecting an increase in DIO2. In a preferred embodiment, the at least one marker gene for decidual senescent cells is DIO2. The method can include detecting and / or quantifying the amounts of both PLA2G2A and DIO2. The method can include detecting a decrease in PLA2G2A and an increase in DIO2. Any of the above genes can be detected and / or quantified in the methods of the invention for assessing the risk of pregnancy loss or embryonic implantation failure, and in the further methods described below.

[0051] Centile or percentile graphs can be used to compare the expression levels of marker genes, such as marker genes for decidual cells and decidual senescent cells (as well as the levels of other markers described below), in samples obtained on different days during the menstrual cycle. Centile graphs are based on the statistical distribution of expression levels of a given marker gene on a given day during the menstrual cycle, for example, after a positive ovulation test. The more samples used to generate the centile graph, the more accurate the reference range. For example, a centile graph may 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 level (i.e., centile) of a given marker gene in an individual's sample obtained on a given day during the menstrual cycle can be calculated relative to the reference percentile graph.

[0052] In some examples, determining the centile for each marker gene makes it possible to determine the cause and clinical symptoms of an individual, such as for recurrent pregnancy loss.The putative defect along the decidual pathway or decidual homeostasis disorder can be determined.For example, when compared with reference sample or reference level, low levels of decidual cell marker genes, such as PLA2G2A, and high levels of decidual senescent cell marker genes, such as DIO2, indicate excessive decidual senescence, which occurs more frequently in recurrent pregnancy loss.In certain examples, when compared with reference sample or reference level, the ratio of PLA2G2A to DIO2 is below the 50th percentile, which indicates a positive diagnosis.In other examples, when compared with reference sample or reference level, the ratio of PLA2G2A to DIO2 is below the 40th percentile, 30th percentile, 20th percentile, or 10th percentile, which indicates a positive diagnosis.

[0053] In another example, the present invention can include determining the relative risk of miscarriage.For example, an individual can be found to be twice as likely to miscarry as to have a live birth.In other embodiments, an individual can be found to be three, four, or five times as likely to miscarry.In a specific example, if the ratio of PLA2G2A to DIO2 is below the 15th percentile, an individual can be found to be twice as likely to miscarry.

[0054] uNK cells and uNK cell gene markers According to the methods of the present invention for assessing the risk of pregnancy loss or implantation failure, in addition to marker genes for decidual cells and decidual senescent cells, typically, the level of uterine natural killer (uNK) cells can also be detected and / or quantified by detection of one or more marker genes for uNK cells.

[0055] Successful endometrial transition (from cyclical tissue to semi-permanent tissue capable of maintaining the placenta throughout pregnancy) relies on stress-resistant decidual cells utilizing uterine natural killer (uNK) cells to eliminate their acutely stressed counterparts, i.e., decidual senescent cells, via granule exocytosis. Thus, it is described herein that the balance of diverging decidual populations and uNK cells during the mid-luteal phase of the menstrual cycle may determine the endometrium's ability to transition to pregnant tissue. An imbalance in decidual subsets is also associated with reproductive failure. Therefore, determining uNK cell levels in combination with detecting marker levels for decidual cells and decidual senescent cells provides additional information for assessing the risk of pregnancy loss or implantation failure, and more generally for diagnosing reproductive disorders.

[0056] The level of uNK cells in a sample can be detected and / or quantified by any means known in the art. uNK cells can be detected and / or quantified using immunohistochemistry and image analysis. Alternatively, uNK cells can be detected based on the level of at least one marker gene for uNK cells in a sample.

[0057] According to the methods of the present invention, any uNK cell gene marker can be detected and / or quantified. 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 for assessing the risk of pregnancy loss or embryonic implantation failure described herein, as well as in the additional methods of the present invention for detecting marker genes described below. Deficiency of uNK cells occurs more frequently in recurrent pregnancy loss.

[0058] Determining timing during the menstrual cycle According to the method of the present invention, the time (for example, time point, stage or day) in the menstrual cycle that sample is obtained can be further determined.Any parameter can be used, including any known hormone, marker or other parameter (including any hormone or marker mentioned above) that allows determining the time point, stage or day in this cycle.Preferably, sample is obtained during embryo implantation window, and the time point, stage or day within the embryo implantation window is determined.By determining the time point, stage or day in the menstrual cycle, it is convenient to compare the sample with the reference sample or level that represents the same time point, stage or day, because hormone level or marker gene level changes throughout the cycle.

[0059] In certain embodiments, therefore, in addition to the marker genes for decidual cells, decidual senescent cells and / or uNK cells, marker genes that allow for the identification of time point, stage or day during the menstrual cycle are also detected and / or quantified.Such marker genes are also referred to herein as molecular timing genes, and typically indicate the timing during the implantation window.Through the analysis of these genes, the accuracy of detection based on the analysis of marker genes for decidual cells and decidual senescent cells and / or uNK cells or uNK cell marker genes can be improved.

[0060] The purpose of molecular timing is twofold. Due to the cycle-dependence of gene marker levels, interpretation of decidual cell gene markers, such as PLA2G2A, decidual senescent cell gene markers, such as DIO2, and / or uNK cell levels or levels of uNK cell gene markers is advantageously aided by knowing the day in the cycle on which the biopsy is performed. In fact, this can also be achieved by scheduling the biopsy in relation to the preovulatory luteinizing hormone (LH) surge, as described above. Thus, the methods of the present invention can be performed in an individual by obtaining a sample at the appropriate time following the LH surge, as described above. However, by considering molecular timing based on analysis of marker gene expression, the risk of mistiming the biopsy due to patient error and inherent variations between the exact time of the LH surge and ovulation can be reduced.

[0061] The implantation window (also known as the receptive window) is associated with dramatic changes in gene expression in the glandular epithelium. Thus, the method of the present invention can preferably include the detection of any marker gene whose expression changes (typically, is selectively expressed) in the glandular epithelium during the implantation window, thereby reporting the time point, stage, or day during the embryo implantation window. The determination can preferably be based on two or more genes that are selectively expressed in the gland and show opposing expression profiles as the menstrual cycle progresses. The ratio of two or more such genes can be determined.

[0062] Marker genes that enable the determination of the molecular timing of the embryo implantation window and can be used in accordance with the present invention include one or more of GPX3, DPP4 (GPX3-like gene), SLC15A2, and CTNNA2 (SLC15A2-like gene).Preferably, the genes that enable the identification of the timing of the day during the menstrual cycle may include GPX3 and SLC15A2, may consist of GPX3 and SLC15A2, or may essentially consist of GPX3 and SLC15A2.Therefore, the ratio of GPX3 to SLC15A2 can be determined.The molecular timing used in the present invention is typically based on genes selectively expressed in epithelial cells, such as GPX3 and SLC15A2.However, since decidual cell markers and decidual senescent cell markers, such as PLA2G2A and DIO2, are selective stromal cell markers, molecular timing can also be used to diagnose the asynchronous hormonal responses in the epithelial and stromal compartments. As described herein, GPX3 and SLC15A2 are regulated in opposing ways as the luteal phase unfolds (i.e., GPX3 is rapidly upregulated, while SLC15A2 is rapidly downregulated).Therefore, the ratio of these two genes varies greatly from day to day during implantation window.The ratio between GPX3 and SLC15A2 is significantly elevated on the LH+5 and LH+11 days of cycle, and thus the ratio between GPX3 and SLC15A2 can be matched with a specific day in cycle.A specific GPX3 / SLC15A2 ratio can be matched with a specific day in cycle based on the centile / percentile graph plotted from the values ​​obtained from the reference sample pooled over that specific day in cycle, for example, as obtained by detecting LH surge (for example, using home ovulation kit).Preferably, if the GPX3 / SLC15A2 ratio in the test sample is between the 25th and 75th percentiles of a reference centile / percentile graph plotted from values ​​obtained from pooled reference samples over a specific day in the cycle and matches (i.e., coincides with) the day of the luteal phase determined by detection of the LH surge (e.g., using a home ovulation kit), the timing of the biopsy can be considered to have improved accuracy, and test results can be reported based on the day in the cycle determined by detection of the LH surge (e.g., using a home ovulation kit). If the GPX3 / SLC15A2 ratio in the test sample falls outside (i.e., does not match) the 25th-75th percentile of a reference centile / percentile graph plotted from values ​​obtained from pooled reference samples over a specific day in the cycle, or if the GPX3 / SLC15A2 ratio does not match the day of the luteal phase determined by detection of the LH surge (e.g., using a home ovulation kit), the timing of the biopsy can be considered less accurate, and test results can be reported both based on the day in the cycle determined by detection of the LH surge using a home ovulation kit, for example, and based on the molecular timing results.

[0063] Marker gene sequence Specific sequences of marker genes useful according to the present invention are disclosed herein, along with their database accession / identification numbers in the NCBI Gene database, the Ensembl database, and the OMIM database. The gene sequences disclosed herein include those available by reference to these online sequence databases as of June 16, 2019. Thus, the following is a list of marker genes with representative accession numbers (in parentheses: NCBI Gene database, followed by the Ensembl database, followed by the OMIM database) and alternative gene names (in italics): Phospholipase A2 group IIA: PLA2G2A (5320, ENSG00000188257, 172411) MOM1, PLA2, PLA2B, PLA2L, PLA2S, PLAS1, sPLA2 Scavenger receptor class A member 5: SCARA5 (286133, ENSG00000168079, 611306) Tesr, NET33, FLJ23907, MGC45780; Ferritin light chain: FTL(2512, ENSG00000087086, 134790)LFTD, NBIA3, MGC71996; Glutaredoxin: GLRX (2745, ENSG00000173221, 600443) GRX, GRX1; Interleukin-1 receptor-like 1: IL1RL1 (9173, ENSG00000115602, 601203) T1, ST2, DER4, ST2L, ST2V, FIT-1, IL33R; Iodothyronine deiodinase 2: DIO2 (1734, ENSG00000211448, 601413) D2, 5DII, SelY, DIOII, TXDI2; Clusterin: CLU (1191, ENSG00000120885, 185430) CLI, AAG4, APOJ, CLU1, CLU2, KUB1, SGP2, APO-J, SGP-2, SP-40, TRPM2, TRPM-2, NA1 / NA2; Insulin-like growth factor binding protein 1: IGFBP1 (3484, ENSG00000146678, 146730) AFBP, IBP1, PP12, IGF-BP25, hIGFBP-1; Glutathione peroxidase 3: GPX3 (2878, ENSG00000211445, 138321) GPx-P, GSHPx-3, GSHPx-P; Solute transporter family 15 member 2: SLC15A2 (6565, ENSG00000163406, 602339) PEPT2; Dipeptidyl-peptidase IV: DPP4 (1803, ENSG00000197635, 102720) CD26, ADABP, ADCP2, DPPIV, TP103; Catenin alpha 2: CTNNA2 (1496, ENSG00000066032, 114025) CAPR, CTNR, CAP-R, CT114, CDCBM9; Interleukin 2 receptor subunit beta: IL2RB (3560, ENSG00000100385, 146710) CD122, IL15RB, P70-75; Interleukin-2 receptor subunit gamma: IL2RG (3561, ENSG00000147168, 308380) P64, CIDX, IMD4, CD132, SCIDX, IL-2RG, SCIDX1; Neural cell adhesion molecule 1: NCAM1 (4684, ENSG00000149294, 116930) CD56, NCAM, MSK39.

[0064] Detecting and / or quantitating the amount / level of biomarkers As used herein, the term "marker gene" or "biomarker" refers to a gene or a fragment of a gene, the change in the amount and / or detection of which can be correlated with a specific physical state or condition. The specific marker gene used in the present invention correlates with the risk of pregnancy loss or embryo implantation failure, and is also used in the methods described herein. The detection and / or quantification of such marker genes can be achieved by any means, and is not limited to the detection / quantification of nucleic acids. Marker genes can also be detected through their respective expression products, such as expressed peptides, polypeptides, and proteins, and fragments thereof.

[0065] As used herein, the term "amount" or "level" refers to the quantity of a marker gene or its expression product that can be detected or measured in a biological sample and / or a control or reference sample. The quantity of a marker gene can be, for example, the quantity of a nucleic acid or protein. The term can alternatively include combinations thereof. The amount or level of a marker gene can refer to the absolute amount or level of a biomarker. Alternatively, a change in the relative level or amount of a marker can be assessed by comparing the level or amount of the marker gene in a sample from a subject with a control or reference value. Alternatively, the relative amount or level of a marker gene can, in some instances, refer to the concentration of the marker gene relative to the total amount or level of the marker gene in a sample.

[0066] The level of a marker gene 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 generate cDNA from the mRNA. The cDNA is used in a qPCR assay to generate fluorescence as the DNA amplification process progresses. By comparison with a standard curve, qPCR can generate absolute measurements, such as the number of mRNA copies per cell. RT-PCR combined with Northern blots, microarrays, Invader assays, and capillary electrophoresis can be used to measure the expression level of mRNA in a sample.

[0067] In some embodiments, polynucleotide biomarkers can be detected using nucleic acid amplification methods. For example, oligonucleotide primers and probes can be used in amplification and detection methods using nucleic acid substrates isolated 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, target 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).

[0068] The detection and quantification of marker genes in the methods of the present invention can also include the use of agents that specifically detect the expression products of marker genes, such as proteins or peptides of interest. The agents can be antibodies or functional equivalents thereof (i.e., anti-peptide antibodies) that bind to proteins or peptides in the analysis. These antibodies can be used to perform immunoassays, such as, but not limited to, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, immunoprecipitation, immunohistochemistry, immunofluorescence, protein dot blots, Western blots, turbidimetry, nephelometry, FACS, etc., which are known to those skilled in the art.

[0069] The relative abundance of marker genes for decidual cells and decidual senescent cells can be expressed as a ratio, for example, 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 the marker gene for decidual cells, and possibly a decrease in the level of the marker gene for decidual senescent cells, will lead to an increase in the ratio. A decrease in the level of the marker gene for decidual cells, and possibly an increase in the level of the marker gene for decidual senescent cells, will lead to a decrease in the ratio.

[0070] How to monitor or evaluate the effectiveness of the treatment The present invention further provides a method for monitoring or evaluating the effectiveness of a treatment for reducing the risk (or likelihood or probability) of pregnancy loss or embryo implantation failure in an individual, 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 the individual, thereby monitoring or evaluating the effectiveness of the treatment, wherein the at least one marker gene for decidual cells comprises PLA2G2A. The correlation between the levels of the marker genes for decidual cells and decidual senescent cells makes it possible to determine the risk of pregnancy loss or implantation failure, as described in the preceding section, thereby determining whether the treatment is effective in reducing the risk. Treatments that can be used to reduce the risk are further described below.

[0071] The marker genes and samples that can be used in the methods for monitoring or assessing the effectiveness of a treatment can be any of those described for use in the preceding sections.

[0072] Monitoring or evaluating the effectiveness of a treatment to reduce the risk of pregnancy loss or embryo implantation failure includes determining whether an individual is responding or has responded to the treatment, determining the nature of the response, determining the degree of response, and determining whether the individual continues to respond to the treatment over time.In some cases, an individual is determined to be responsive to the treatment or to have had a positive response.Responsiveness or a positive response to a treatment means that the individual is expected to benefit or benefit to a sufficient degree as a result of the treatment.For example, the individual may have or be expected to have a successful pregnancy, or the individual may have an improved prognosis.Non-responsiveness or a negative response to a treatment means that the individual is not expected to benefit or benefit to a sufficient degree from receiving the treatment.

[0073] An increased or increased level of a marker gene for decidual cells, such as PLA2G2A, in a sample compared to a reference sample or level can indicate a positive response to treatment. Alternatively, a decreased or decreased level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can indicate a positive response to treatment. For example, an increased or increased level of a marker gene for decidual cells, such as PLA2G2A, and a decreased or decreased level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can both 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, for example, the PLA2G2A / DIO2 ratio. The fold change in this ratio can indicate whether the treatment is effective. For example, an increase in the level of the marker gene for decidual cells, and possibly a decrease in the level of the marker gene for decidual senescent cells, would lead to an increase in the ratio, indicating a positive response to treatment by attenuating decidual senescence.

[0074] A decreased, decreased, or unchanged level of a marker gene for decidual cells, such as PLA2G2A, in a sample compared to a reference sample or level can indicate a negative response to treatment. Alternatively, an increased, increased, or unchanged level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can indicate a negative response to treatment. For example, a decreased, decreased, or unchanged level of a marker gene for decidual cells, such as PLA2G2A, and an increased, increased, or unchanged level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can both indicate a negative response to treatment. A decrease in the level of the marker gene for decidual cells, and possibly an increase in the level of the marker gene for decidual senescent cells, can cause a decrease in the ratio of the marker gene for decidual cells to the marker gene for decidual senescent cells, which indicates a negative response to treatment.

[0075] As an example, a method for monitoring or assessing the effectiveness of a treatment for reducing the risk of pregnancy loss or embryonic 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 the sample. For example, detecting and / or quantifying the level of uNK cells based on the marker gene can be as described in the preceding section.

[0076] Increased or increasing levels of uNK cells or uNK cell gene markers in the sample compared to the reference sample can indicate a positive response to the treatment. Decreased or decreasing levels of uNK cells or uNK cell gene markers compared to the reference sample can indicate a negative response to the treatment.

[0077] The above methods of treatment of the present invention may also further comprise the step of detecting and / or quantifying genes that allow identification of the stage, time point or day of the menstrual cycle as described in the preceding section.

[0078] Control sample or reference sample or level can be selected according to any of the above criteria.In the method for monitoring or evaluating the effect of treatment for reducing the risk of pregnancy loss or embryo implantation failure in individuals, the level of marker gene (for example, PLA2G2A and DIO2) at the first time point before treatment can be compared with the level of marker gene (for example, PLA2G2A and DIO2) at a later time point during or after treatment.The level of marker gene (for example, PLA2G2A and DIO2) during treatment can also be compared with the level of marker gene (for example, PLA2G2A and DIO2) at a later time point during or after treatment.In some examples, the level of marker gene can be determined monthly, every other month, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or any other suitable time interval determined by a physician.

[0079] In an aspect related to the above method, the present invention also provides a method for preventing or reducing the risk of pregnancy loss or implantation failure in an individual, wherein at least one marker gene for decidual cells comprises PLA2G2A, the method comprising the steps of 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 administering an agent or treatment regimen effective for preventing or reducing the risk of pregnancy loss or implantation failure in the individual. As described above, if the marker gene level indicates a risk of pregnancy loss or implantation failure, the agent is administered or the treatment regimen is carried out.

[0080] How to Diagnose Reproductive Disorders The present invention further provides a method for diagnosing a reproductive disorder in an individual, 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 the individual, thereby diagnosing the disorder, wherein the at least one marker gene for decidual cells comprises PLA2G2A. The marker genes that can be used in the method for diagnosing a reproductive disorder are as described in the preceding section.

[0081] The reproductive disorder can be any reproductive disorder. The reproductive disorder can be any disorder associated with infertility, miscarriage, the risk of labor complications, or that negatively impacts pregnancy outcome. The reproductive disorder described herein can be any disorder involving reduced receptivity or the inability of the endometrium to accept an embryo. Such disorders can include embryo implantation failure, miscarriage, recurrent pregnancy loss, or placental disorders. In a preferred embodiment, the reproductive disorder is recurrent pregnancy loss.

[0082] Diagnosis involves determining whether an individual has a reproductive disorder. Diagnosis can also involve determining the specific cause of the reproductive disorder and assessing various clinical symptoms. A positive diagnosis involves a determination that an individual has the disorder. A negative diagnosis involves a determination that an individual does not have the disorder.

[0083] A decreased or decreased level of a marker gene for decidual cells, such as PLA2G2A, in a sample compared to a reference sample or level can indicate a positive diagnosis. Alternatively, an increased or increased level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can indicate a positive diagnosis. For example, a decreased or decreased level of a marker gene for decidual cells, such as PLA2G2A, and an increased or increased level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can both indicate a positive diagnosis.

[0084] A constant (or similar), increased or increasing level of a marker gene for decidual cells, such as PLA2G2A, in a sample compared to a reference sample or level can indicate a negative diagnosis. Alternatively, a constant (or similar), decreased or decreasing level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can indicate a negative diagnosis. For example, a constant (or similar), increased or increasing level of a marker gene for decidual cells, such as PLA2G2A, and a constant (or similar), decreased or decreasing level of a marker gene for decidual senescent cells, such as DIO2, in a sample compared to a reference sample or level can both indicate a negative diagnosis.

[0085] As an example, the method for diagnosing a reproductive disorder in an individual further includes 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 method of diagnosis can be as described in the preceding section.

[0086] 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.

[0087] The diagnostic method of the present invention may further comprise the step of detecting and / or quantifying genes that allow identification of the day in the menstrual cycle as described in the previous section.

[0088] Control or reference samples or levels can be provided according to the above criteria and can represent levels from one or more individuals known to have a reproductive disorder or known not to have any reproductive disorder.

[0089] Therapeutic methods The present invention also provides a method for treating a reproductive disorder in an individual. The method includes diagnosing the reproductive disorder according to the method described in the preceding section, and administering an agent or treatment regimen effective for treating the reproductive disorder to a positively diagnosed individual. Agents for use in the method for treating a reproductive disorder in an individual are also described, wherein the reproductive disorder is diagnosed according to the method described in the preceding section. Use of the agents for preparing a medicament for treating a reproductive disorder is also described. In some embodiments, the individual has an increased level of at least one marker gene, such as DIO2, in decidual senescent cells. In some embodiments, the individual has a decreased level of at least one marker gene, such as PLA2G2A, in decidual cells. The term "treating" includes reducing or preventing the onset 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 the marker level changes, resulting in a negative diagnosis. For example, the relative abundance of marker genes in decidual cells and decidual senescent cells can be expressed as a ratio, such as the PLA2G2A / DIO2 ratio. The fold change in this ratio can indicate whether the treatment is effective. For example, an increase in the level of the marker gene for decidual cells, and possibly a decrease in the level of the marker gene for decidual senescent cells, would lead to an increase in the ratio, indicating a positive response to the treatment due to the attenuation of decidual senescence. There may be a decrease in the ratio of the marker gene for decidual cells to the marker gene for decidual senescent cells, caused by a decrease in the level of the marker gene for decidual cells, and possibly an increase in the level of the marker gene for decidual senescent cells, indicating a negative response to the treatment.

[0090] The drug or treatment regimen that can be administered or performed can be any drug or treatment regimen known to be effective in treating reproductive disorders.The drug or treatment regimen can be any that can increase the level of decidual cells and / or uNK cells and / or reduce the level of decidual senescent cells in an individual.Suitable drug or treatment regimens include, but are not limited to, endometrial scratch, dipeptidyl peptidase IV (DPP4) inhibitors (typically gliptins, such as sitagliptin), and senolytic drugs (e.g., dasatinib, quercetin). Examples of DPP4 inhibitors include vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin, evogliptin, omarigliptin, and teneligliptin, as described in, for example, Deacon CF & Lebovitz HE, Diabetes Obes Metab., 2016;18(4):333-47. Drugs or treatment regimens can target different types of decidual homeostasis dysregulation, as determined based on the aforementioned diagnostic methods. For example, endometrial scratching can be used to treat decidual insufficiency. Senolytic drugs can be used to treat age-related reproductive disorders. Senolytic drugs or senolytic agents are drugs that can target cellular senescence to delay, prevent, alleviate, or reverse age-related disorders. The above-mentioned drugs and treatment regimens are also described for use in methods for reducing or preventing the risk of pregnancy loss or embryo implantation failure.

[0091] In a preferred embodiment, the agent is a DPP4 inhibitor or antagonist. DPP4 is a known marker of glandular differentiation during the midluteal phase of the cycle and is a ubiquitous aminopeptidase expressed both as a cell surface-associated protein and in a soluble form (59, 60). DPP4 is also a widely used endometrial receptivity marker gene (61). Stromal cell-derived factor 1α (SDF-1), also known as C-X-C motif chemokine ligand 12 (CXCL12), is a potent chemotactic factor that mediates BMDC recruitment and homing to the endometrium in response to tissue injury and elevated estradiol levels (62, 63). However, SDF-1 is proteolytically inactivated by DPP4. We have determined that DPP4 inhibitors (gliptins), oral antidiabetic drugs commonly used to treat type 2 diabetes (64), can be used to reduce excessive decidual senescence in RPL patients by increasing endometrial stem cells or inhibiting the expression of marker genes for senescent decidual cells, such as DIO2.

[0092] A DPP4 inhibitor or antagonist can be any agent that inhibits or antagonizes the expression or activity of DPP4 by any means. The agent can inhibit or antagonize the inactivation of SDF-1 by DPP4. Such agents can be small molecules, peptides, proteins, antibodies, polynucleotides, oligonucleotides, antisense RNA, small interfering RNA (siRNA) or short hairpin RNA (shRNA), or any other suitable inhibitors that achieve the above functions. The agent can be a polynucleotide that encodes a molecule that inhibits or antagonizes DPP4, or typically a polynucleotide, oligonucleotide, antisense RNA, siRNA, or shRNA that contains a complementary sequence to DPP4 mRNA and specifically hybridizes to DPP4 mRNA, inhibiting the expression of DPP4. An oligonucleotide "specifically hybridizes" to a target sequence if it hybridizes preferentially or with high affinity to the target sequence, but does not substantially hybridize, does not hybridize, or only hybridizes with low affinity to other sequences. More preferably, the oligonucleotide has a T domain relative to another nucleic acid. m at least 5°C, at least 10°C, at least 20°C, at least 30°C, or at least 40°C higher than T m The hybridization condition can be stringent as described in the art. The hybridization condition is well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory 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)). The hybridization condition can be stringent as described in the art.

[0093] The agent can be an antibody that specifically binds to DPP4 protein or another protein that indirectly inhibits DPP4 function.An antibody "specifically binds" to a protein if it binds to that protein preferentially or with high affinity, but does not substantially bind, does not bind, or only binds with low affinity to other proteins.For example, an antibody "specifically binds" to a target molecule if it binds to its target preferentially or with high affinity, but does not substantially bind, does not bind, or only binds with low affinity to other human proteins.

[0094] The antibody is 1 x 10 -7 M or less, preferably 5×10 -8 M or less, preferably 1×10 -8 M or less, or more preferably 5 × 10 -9 An antibody binds with preferential or high affinity if it binds with a Kd of 1×10 M or less. -6 M or more, preferably 1×10 -5 M or more, preferably 1×10 -4 M or more, preferably 1×10 -3 M or more, and even more preferably 1×10 -2 If it binds with a Kd of M or greater, it binds with low affinity.

[0095] The antibody may 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 may be an intact immunoglobulin molecule or a fragment thereof, such as a Fab, F(ab')2, or Fv fragment.

[0096] In a preferred embodiment, the agent used in the method of treatment may be a gliptin, including, for example, sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin, or dutogliptin. Preferably, the gliptin is sitagliptin.

[0097] In some embodiments, the treatment can include detecting an increased level of a marker gene for decidual senescent cells (e.g., DIO2). If an increase in DIO2 is detected, the agent used is preferably a DPP4 inhibitor, typically gliptin, more preferably sitagliptin.

[0098] The specific route, dosage and method of administration of the therapeutic agent described herein can be routinely determined by a physician.The drugs used in the treatment methods described herein can be formulated into pharmaceutical compositions.These compositions can contain, in addition to the therapeutically active ingredient, pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers or other materials known to those skilled in the art.Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient.The pharmaceutical carrier or diluent can be, for example, an isotonic solution.

[0099] The dosage can be determined according to various parameters, in particular according to the agent used, the age, weight, and condition of the patient to be treated, the route of administration, and the required regimen. Again, the physician can determine the route of administration and dosage required for any particular patient.

[0100] Drug can be administered to patient by any suitable means.Drug can be administered by enteral or parenteral route, such as oral, buccal, anal, pulmonary, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraarticular, topical or other suitable administration route.For example, when drug is DPP4 inhibitor, such as sitagliptin, it is preferably administered orally.

[0101] The daily dosage of a gliptin, e.g., sitagliptin, for administration to a subject, e.g., a human, is about 50 mg / day to about 2000 mg / day, e.g., about 50 mg / day to about 1500 mg / day, about 50 mg / day to about 100 mg / day, about 75 mg / day to about 150 mg / day, about 100 mg / day to about 1500 mg / day, about 100 mg / day to about 1200 mg / day, about 100 mg / day to about 175 mg / day, about 150 mg / day to about 300 mg / day, about 2 The daily dose of sitagliptin can range from about 100 mg / day to about 350 mg / day, about 250 mg / day to about 400 mg / day, about 300 mg / day to about 450 mg / day, about 350 mg / day to about 500 mg / day, about 400 mg / day to about 550 mg / day, about 450 mg / day to about 600 mg / day, about 500 mg / day to about 750 mg / day, about 600 mg / day to about 800 mg / day, about 700 mg / day to about 1000 mg / day, or about 800 mg / day to about 1200 mg / day. Preferably, a typical daily dose of sitagliptin is about or at least about 100 mg / day.

[0102] Administration can be in a single or multiple doses. Multiple doses can be administered via the same or different routes and to the same or different locations. Alternatively, administration can be via sustained-release formulations, in which case less frequent administration is required. The dosage and frequency can vary depending on the half-life of the drug in the patient and the desired duration of treatment. The dosage can be administered once a day or divided into two doses. The drug can be administered more than once, for example, for at least two or at least three consecutive menstrual cycles. For example, a 100 mg sitagliptin capsule can be given orally once a day for two or three consecutive menstrual cycles.

[0103] Methods of treatment for medical use can include administering to the individual an additional agent known to be effective in treating reproductive disorders, for example, progesterone and / or a progestogen can be further administered.

[0104] Also provided herein is a method 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 drug or treatment regimen effective for preventing pregnancy loss or embryo implantation failure, wherein the at least one marker gene for decidual cells comprises PLA2G2A. The drug or treatment regimen can be any of the above-mentioned drugs or treatment regimens, preferably administering a gliptin, such as sitagliptin.

[0105] Also provided is a method for assessing readiness for conception or the success of embryo implantation.Such method also includes 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 readiness for conception or embryo implantation, wherein at least one marker gene for decidual cells comprises PLA2G2A.The marker gene and detection method that can be used in the above method are as described in the preceding section.

[0106] Methods for selecting patients for treatment The present invention describes a method for selecting patients for treatment to reduce the risk (or likelihood or probability) of embryo implantation failure or pregnancy loss in an individual, 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 the individual, thereby selecting patients for treatment to reduce the risk of pregnancy loss or embryo implantation failure based on the level of the marker genes, wherein the at least one marker gene for decidual cells includes PLA2G2A. The method may include detecting and / or quantifying the level of uNK cells in the sample or a uNK cell gene marker in the sample. Marker genes that can be used in the patient selection method are similar to those described in the preceding section. The treatment may employ any of the treatment regimens or agents described above.

[0107] Individuals in which an increased level of at least one marker gene for decidual senescent cells, such as DIO2, is detected can be selected as patients for treatment. Individuals in which a decreased level of at least one marker gene for decidual cells, such as PLA2G2A, is detected can also be selected as patients for 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.

[0108] The method of the present invention may further comprise the step of detecting and / or quantifying genes that allow identification of the days in the menstrual cycle as described in the previous section.

[0109] The correlation between the levels of marker genes for decidual cells, decidual senescent cells, and uNK cells may allow for the determination of specific defects in the decidual pathway, which may then allow for the determination of whether a patient may benefit from selecting a specific type of treatment to reduce the risk of embryo implantation failure or miscarriage.

[0110] How to stratify patients The present invention further provides a method for stratifying patients into different groups, for example, for clinical research. The method includes 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, where the at least one marker gene for decidual cells includes PLA2G2A. The method can include, for example, detecting and / or quantifying the level of uNK cells in the sample based on the level of a uNK cell gene marker in the sample. The marker genes that can be used in the method for stratifying patients can be any of those described in the preceding sections.

[0111] The method of the present invention may further comprise the step of detecting and / or quantifying genes that allow identification of the days in the menstrual cycle as described in the previous section.

[0112] The correlation between the levels of marker genes for decidual cells, decidual senescent cells, and uNK cells may allow the identification of specific defects in the decidual pathway, and patients with different patterns or levels of these markers can then be appropriately grouped for clinical studies.

[0113] kit The present invention further provides a kit suitable for use in any of the methods of the present invention. The kit may include means (e.g., reagents) for detecting and / or quantifying at least one marker gene for decidual cells and at least one marker gene for decidual senescent cells at the nucleic acid or protein level in a biological sample from an individual, where the at least one marker gene for decidual cells includes PLA2G2A. Thus, the kit includes a reagent for detecting and / or quantifying PLA2G2A. The at least one marker gene for decidual senescent cells may include DIO2. The at least one marker gene for decidual senescent cells may be selected from DIO2, CLU, and IGFBP1. The kit may also include means for detecting and / or quantifying the level of uNK cells or uNK cell markers. Marker genes that can be detected using the kit may be any of those described in the preceding sections. The kit may also include means for detecting and / or quantifying genes that enable identification of days in the menstrual cycle, as described in the preceding sections. Preferably, the genes that allow for the identification of the timing of the days in the menstrual cycle comprise, consist or consist essentially of GPX3 and SLC15A2.

[0114] The only reagents included in the kit for detecting and / or quantifying marker genes may be the reagents for detection of the marker genes specified above.

[0115] The kit may further comprise instructions for using the kit in accordance with the methods of the invention. The kit may also include details regarding on which individuals the method can be performed. The kit may also be provided with means for obtaining an endometrial biopsy sample. The kit may also include a test requisition form with details to be sent to the analyst. The kit may further comprise means for measuring other tests or clinical parameters, and / or a container for holding a biological sample isolated from the subject.

[0116] The kit may further comprise one or more other reagents or devices that enable the method to be carried out. Such reagents or devices may include one or more of the following: appropriate buffers (aqueous solutions), calibration standards, color reagents, enzymes, labels, reaction surfaces, means for detection, control samples, standards, instructions, interpretive information, means for isolating relevant biomarkers from a sample, means for obtaining a sample from an individual (e.g., a container or device containing a needle), or a support containing a well in which a quantitative reaction can be carried out.

[0117] As an example, the kit can include cryotubes and an RNA stabilizing solution.

[0118] As an example, the use of the above test kit is described for assessing pregnancy loss or embryo implantation failure, or for diagnosing reproductive disorders.The use can include the steps described above in connection with the method of the present invention for detecting marker genes.In a preferred embodiment, the kit is used to assess the risk of recurrent pregnancy loss or for diagnosing recurrent pregnancy loss. [Example]

[0119] material and method Ethics approval and sample collection 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 at the University Hospitals of Coventry and Warwickshire (UHCW) National Health Service Trust. Excess tissue from endometrial biopsies obtained for diagnostic purposes at the Implantation Research Clinic was used in this study. Samples were obtained during the luteal phase of ovulatory, non-hormonally stimulated menstrual cycles, timed to the preovulatory LH surge, using a Wallach Endocell™ endometrial sampler. Overt uterine pathology was excluded by transvaginal ultrasound scan prior to biopsy.

[0120] Drop-Seq analysis of timed endometrial biopsies Six LH-timed endometrial biopsies were processed as described in detail elsewhere (Lucas et al., 2020). After tissue digestion, red blood cells were removed from the flow-through by Ficoll density gradient centrifugation. The single-cell fraction was then subjected to Drop-Seq analysis.

[0121] Reverse transcription quantitative PCR (RT-qPCR) RNA was extracted from endometrial biopsies submitted to RNA lattice in 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 an equivalent volume of 10 ng / μl before use in qPCR. Amplification was performed in a QuantStudio 5 (ThermoFisher) in 10 μl reactions using 2x Quantifast SYBR Green RT-PCR Kit (QIAGEN) with 300 nM each of forward and reverse primers. Primer sequences were as follows: DIO2 forward: 5'-ACT CGG TCA TTC TGC TCA A-3', DIO2 reverse: 5'-TTC CAG ACG CAG CGC AGT-3', PLA2G2A forward 5'AAA GGA AGC CGC ACT CAG TT-3', PLA2G2A reverse: 5'-TTT CCA GGG AAG AGG GGA C-3'. Centile calculations were performed on dCt values ​​using R v3.5 software.

[0122] Multiplexed single molecule in situ hybridization (RNAScope) Formalin-fixed, paraffin-embedded (FFPE) samples were cut into 5 μm sections. RNA in situ hybridization was performed with the RNAscope® 2.5HD Duplex Reagent Kit (ACD, California, USA) using probes for PLA2G2A (581101-C2) and DIO2 (562211) according to the manufacturer's guidelines. Following 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.

[0123] Spatial transcriptomics Endometrial biopsies were fixed overnight in 10% neutral buffered formalin at 4°C and wax-embedded in Surgipath Formula "R" paraffin using a Shandon Excelsior ES Tissue processor (ThermoFisher). Four endometrial biopsies were selected based on morphology, PLA2G2A / DIO2 expression, and RNA integrity (DV.200 > 50). Five-micron sections were prepared, deparaffinized, and stained with hematoxylin and eosin according to a protocol (10xGenomics). Spatial gene expression slides and reagent kits were used according to the manufacturer's instructions. Each capture area (6.5 x 6.5 mm) contained 5,000 barcoded spots with a 55-micron diameter (100-micron center-to-center spacing between spots, yielding an average resolution of 1–10 cells). Eluted libraries were analyzed using an Agilent Bioanalyzer High Sensitivity DNA chip to assess quality and determine library size. Libraries were diluted and denatured using standard Illumina protocols and sequenced using the NextSeq 500 / 550 High Output Kit v2.5 (150 cycles). Sequencing was performed using the recommended 10X protocol (Read 1: 28 cycles; i7 Index Read: 10 cycles; i5 Index Read: 10 cycles; and Read 2: 91 cycles), yielding 21–50 million sequence reads. Reads were processed using Spaceranger software v1.3.0, using refdata-gex-GRCh38-2020-A as the reference genome data, and analyzed in R v4.1.3 using Seurat v4.0.4.

[0124] Bulk RNA sequencing Total RNA was extracted from endometrial biopsies using the RNeasy Plus Universal Mini Kit (QIAGEN) according to the manufacturer's instructions. Libraries were diluted and denatured using standard Illumina protocols and sequenced using the NextSeq 500 / 550 High Output Kit v2.5 (75 cycles). Reads were mapped to the GRCh38 human genome assembly and gencode v38 annotation using STAR v2.7.9. Reads overlapping genic 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.

[0125] SIMPLANT Clinical Trial Data Details regarding the methods and governance of the SIMPLANT clinical trial can be found in Tewary et al., 2020. Briefly, a double-blind, randomized, placebo-controlled feasibility study was conducted in women aged 18-42 years with a history of three or more miscarriages. Thirty-eight subjects were randomized to either the DPP4 inhibitor sitagliptin (100 mg daily) or identical placebo capsules for three consecutive cycles. Tissue samples were used for exploratory studies.

[0126] result 1. Discovery of a novel biomarker for anti-inflammatory decidual cells Using high-throughput single-cell droplet barcoding of transcriptome changes along the decidual pathway in vitro, we defined the characteristics of divergent decidual subpopulations. Single-cell RNA-sequencing analysis of luteal phase endometrial biopsies first identified two putative biomarkers, SCARA5 and DIO2, as selective marker genes for decidual cells and stressed / senescent cells, respectively (Lucas et al., 2020; WO 2021 / 032973).

[0127] Further bulk RNA sequencing (RNA-seq) of paired biopsies from the same patient but from different cycles uncovered the biomarker gene PLA2G2A, encoding phospholipase A2 group IIA (Figures 2A and 2B). Similar to SCARA5, PLA2G2A is a stromal cell-specific biomarker gene for progesterone-dependent decidual cells, but it has a much larger dynamic range of expression levels, making it a significantly more sensitive biomarker. Both DIO2 and PLA2G2A are highly abundant in the endometrial stroma (Figure 2C), but they show contrasting temporal regulation throughout the menstrual cycle (Figure 2D). We generated reference ranges for the expression of both genes in the peri-implantation endometrium by RTQ-PCR analysis of 822 biopsy samples obtained on days 6–11 of the preovulatory luteinizing hormone (LH+6–LH+11) surge as determined by a nonprescription home ovulation test kit (Figure 2E). Percentiles are used to compare the relative expression of biomarkers in endometrial samples obtained on different days during the menstrual cycle.

[0128] We elucidated the spatial organization of PLA2G2A- and DIO2-expressing stromal subpopulations using multiplex single-molecule in situ hybridization (RNAScope®) and Visium spatial transcriptomics (10xGenomics). Spatial analysis confirmed that DIO2 and PLA2G2A mark distinct stromal subpopulations in the peri-implantation endometrium (Figure 3). Furthermore, DIO2-positive cells were abundant near the luminal epithelium (lining the uterine cavity), whereas PLA2G2A-positive cells were present deeper in the tissue. Thus, quantification of DIO2 and PLA2G2A transcript levels in endometrial samples provides information about the tissue spatial organization during the peri-implantation window.

[0129] 2. Diagnostic and prognostic value of the normalized PLA2G2A / DIO2 expression ratio in luteal phase endometrial biopsies in recurrent pregnancy loss We analyzed the PLA2G2A / DIO2 ratio (expressed as a percentile of the ratio) in 854 LH-timed endometrial biopsies from women with 0 to 18 previous miscarriages. The lower the ratio of these marker genes, the higher the relative excess of stressed / senescent cells over anti-inflammatory decidual cells in the sample, and vice versa. As shown in Figure 4A, the median PLA2G2A / DIO2 ratio gradually decreased in this sample set as a function of the number of previous miscarriages. The frequency of samples with ratios below the 25th percentile (lower quartile) decreased, while the frequency of samples with ratios above the 75th percentile (upper quartile) gradually increased with each additional loss (Figure 4B). A cutoff PLA2G2A / DIO2 ratio of <15 percentile maximized separation between samples from women with no history of previous pregnancy loss and those with six or more previous miscarriages (Figure 4C). Importantly, neither maternal age nor timing of biopsy relative to the preovulatory LH surge differed significantly between samples below or above the 15th percentile cutoff ratio (Figure 4D). However, patients below the 5th percentile cutoff ratio had a significantly higher body mass index (BMI) (P<0.001), a well-documented risk factor for recurrent miscarriage / recurrent pregnancy loss (Quenby et al., 2021).

[0130] We also analyzed 217 endometrial biopsies obtained before subsequent pregnancies. No time interval between the date of biopsy and the onset of pregnancy was restricted. As shown in Figure 5A, recurrent miscarriage patients with a low PLA2G2A / DIO2 ratio were significantly more likely to experience a second miscarriage (<20th percentile). Conversely, patients with a high PLA2G2A / DIO2 ratio (>60th percentile) were less likely to experience a second miscarriage. Importantly, maternal age, body mass index (BMI), and uNK cell levels did not significantly differ between patients who had a live birth or pregnancy loss after endometrial biopsy (P > 0.05) (Figure 5B).

[0131] 3. Therapeutic intervention: Sitagliptin treatment improves the PLA2G2A / DIO2 expression ratio in recurrent pregnancy loss We previously reported that sitagliptin, a dipeptidyl peptidase IV (DPP4) inhibitor used in the management of type 2 diabetes, increases the mobilization of bone marrow-derived MSCs when given over three menstrual cycles (Tewary et al., 2020). Using endometrial samples obtained during this double-blind, placebo-controlled, randomized pilot study, we examined the effects of sitagliptin compared with placebo on the endometrial PLA2G2A / DIO2 ratio before and after the study. As shown in Figure 6, patients receiving sitagliptin had a significant improvement in the endometrial PLA2G2A / DIO2 ratio (P = 0.0052). In contrast, in the placebo group, the endometrial PLA2G2A / DIO2 ratio did not differ significantly between before and after the study (P > 0.05).

[0132] References

[0133] [Table 1-1]

[0134] [Table 1-2]

[0135] [Table 1-3]

[0136] [Table 1-4]

[0137] [Table 1-5]

[0138] [Table 1-6]

[0139] Embodiment 1. A method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, comprising the step of 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 of embodiment 1, wherein the at least one marker gene for 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). 3. The method of any one of the preceding embodiments, wherein the at least one marker gene for decidual senescent cells is selected from iodothyronine deiodinase 2 (DIO2), clusterin (CLU) and insulin-like growth factor binding protein 1 (IGFBP1). 4. The method of embodiment 3, wherein the at least one marker gene for decidual senescent cells is DIO2. 5. The method of any one of the preceding embodiments, comprising detecting and / or quantifying the amount of PLA2G2A and DIO2. 6. Decreased levels of marker genes for decidual cells compared to a reference sample or level, and / or an increased level of a marker gene for decidual senescent cells compared to a reference sample or level; 10. The method of any one of the preceding embodiments, wherein the individual is indicated to be at risk for pregnancy loss or embryo implantation failure. 7. The method of 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. 8. The method of embodiment 7, wherein a decreased level of uNK cells or a decreased level of at least one marker gene for uNK cells, as compared to a reference sample or level, indicates that the individual is at risk of pregnancy loss or embryonic implantation failure. 9. The method of any one of the preceding embodiments, further comprising determining one or more risk indicators selected from the group consisting of maternal body mass index, maternal age, and number of previous pregnancy losses or embryo implantation failures. 10. A method for monitoring or evaluating the effectiveness of a treatment for reducing the risk of pregnancy loss or embryonic implantation failure in an individual, comprising the step of 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 monitoring or evaluating the effectiveness of the treatment, wherein the at least one marker gene for decidual cells comprises PLA2G2A. 11. The method of embodiment 10, wherein the at least one marker gene for decidual cells further comprises SCARA5, FTL, GLRX and / or IL1RL1. 12. The method of embodiment 10 or 11, wherein the at least one marker gene for decidual senescent cells is selected from DIO2, CLU and IGFBP1. 13. The method of embodiment 12, wherein at least one marker gene for decidual senescent cells is DIO2. 14. The method of any one of embodiments 10 to 13, comprising detecting and / or quantifying the amount of PLA2G2A and DIO2. 15. (i) An increased level of the marker gene for decidual cells compared to a reference sample or level, and / or a decreased level of the marker gene for decidual senescent cells compared to a reference sample or level indicates a positive response to the treatment; (ii) The method of any one of embodiments 10-14, wherein a decreased level of the marker gene for decidual cells compared to a reference sample or level, and / or an increased level of the marker gene for decidual senescent cells compared to a reference sample or level, indicates a negative response to the treatment. 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. 17. The method of embodiment 16, wherein (i) an increased level of uNK cells or 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 decreased level of uNK cells or at least one marker gene for uNK cells compared to a reference sample or level indicates a negative response to the treatment. 18. The method of any one of embodiments 10-17, comprising comparing the level of the marker gene at a first time point before or during treatment with the level of the marker gene at a subsequent time point during or after treatment. 19. The method of any one of the preceding embodiments, wherein the risk of pregnancy loss is a risk of euploid pregnancy loss or the risk of embryo implantation failure is not due to a chromosomal abnormality in the embryo. 20. The method of any one of the preceding embodiments, wherein the risk of pregnancy loss is a risk of recurrent pregnancy loss. 21. A method for diagnosing a reproductive disorder in an individual, 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 the individual, thereby diagnosing the disorder, wherein the at least one marker for decidual cells comprises PLA2G2A. 22. The method of embodiment 21, wherein the reproductive disorder is embryonic implantation failure, pregnancy loss, recurrent miscarriage, recurrent pregnancy loss or placental disorder. 23. The method of embodiment 21 or embodiment 22, wherein the at least one marker gene for decidual cells further comprises SCARA5, FTL, GLRX and / or IL1RL1. 24. The method according to any one of embodiments 21 to 23, wherein the at least one marker gene for decidual senescent cells is selected from DIO2, CLU and IGFBP1. 25. The method of embodiment 24, wherein at least one marker gene for decidual senescent cells is DIO2. 26. The method according to any one of embodiments 21 to 25, comprising detecting and / or quantifying the amount of PLA2G2A and DIO2. 27. Decreased levels of decidual cell marker genes when compared to a reference sample or level, and / or an increased level of the decidual senescent cell marker gene as compared to a reference sample or level; The method of any one of embodiments 21 to 26, which indicates a positive diagnosis. 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. 29. The method of embodiment 28, wherein a decreased level of uNK cells or at least one marker gene for uNK cells, as compared to a reference sample or level, indicates a positive diagnosis. 30. In a positive diagnosis, the ratio of PLA2G2A to DIO2 is: (i) below the 50th percentile when compared to a reference sample or reference level; or (ii) below the 30th percentile when compared to a reference sample or reference level; 30. The method according to any one of embodiments 27 to 29. 31. The method of any one of the preceding embodiments, wherein the biological sample is an endometrial biopsy sample. 32. The method of any one of the preceding embodiments, wherein the biological sample is collected during the luteal phase of the menstrual cycle, and optionally, the biological sample is collected during the mid-luteal phase of the menstrual cycle. 33. The method of any one of the preceding embodiments, wherein the individual is suffering from or has suffered from infertility or embryo implantation failure after an IVF procedure. 34. The method of any one of the preceding embodiments, wherein the individual has already suffered at least one previous pregnancy loss or embryo implantation failure, or is suffering from recurrent pregnancy loss. 35. The method of any one of the preceding embodiments, further comprising detecting and / or quantifying genes that allow identification of the day in the menstrual cycle, and optionally wherein the genes that allow identification of the timing of the day in the menstrual cycle comprise, consist of, or consist essentially of glutathione peroxidase 3 (GPX3) and solute transporter family 15 member 2 (SLC15A2). 36. The method of any one of the preceding embodiments, wherein the marker genes are detected and / or quantified using ELISA, Western blotting, immunohistochemistry, immunoassay, enzymatic assay or sequencing methods, and optionally the sequencing methods comprise qPCR, Taqman-PCR, multiplex Taqman-PCR, Nanostring, targeted sequencing or digital PCR. 37. The method of embodiment 36, wherein the digital PCR is digital droplet PCR (ddPCR). 38. A method for treating a reproductive disorder in an individual, comprising diagnosing the reproductive disorder according to a method according to any one of embodiments 21 to 37, and administering, in a positively diagnosed individual, an agent or treatment regimen effective to treat the reproductive disorder. 39. The method of embodiment 38, wherein the agent or treatment regimen increases the level of decidual cells and / or uNK cells, and / or decreases the level of decidual senescent cells in the individual. 40. The method of embodiment 38 or 39, wherein the agent is a DPP4 inhibitor. 41. The method of embodiment 40, wherein the DPP4 inhibitor is sitagliptin. 42. The method of any one of embodiments 38-41, wherein the individual has an increased level of at least one marker gene for decidual senescent cells. 43. The method of any one of embodiments 38 to 42, wherein at least one marker gene for decidual senescent cells is DIO2. 44. The method of any one of embodiments 38-43, comprising administering progesterone and / or a progestogen. 45. The method of any one of embodiments 21-44, wherein the reproductive disorder is recurrent pregnancy loss. 46. ​​A method for selecting a patient for treatment to reduce the risk of embryo implantation failure or pregnancy loss, 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 the 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 for decidual cells comprises PLA2G2A. 47. The method of embodiment 46, wherein individuals in whom an increased level of at least one marker gene for decidual senescent cells is detected are selected for treatment. 48. The method of embodiment 47, wherein the patient is selected for treatment with a DPP4 inhibitor. 49. The method of embodiment 48, wherein the DPP4 inhibitor is sitagliptin. 50. The method of any one of embodiments 46 to 49, wherein at least one marker gene for decidual senescent cells is DIO2. 51. The method of embodiments 46-50, wherein the method of selecting patients for treatment is to reduce the risk of pregnancy loss, and the pregnancy loss is recurrent pregnancy loss. 52. A test kit suitable for use in the method of any one of the preceding embodiments, comprising means 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 means for detecting and / or quantifying in the individual the level of uNK cells or the level of at least one marker gene for uNK cells, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

Claims

1. 1. A method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, comprising the step of 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. (a) the at least one marker gene for 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 for decidual senescent cells is selected from iodothyronine deiodinase 2 (DIO2), clusterin (CLU) and insulin-like growth factor binding protein 1 (IGFBP1), and optionally the at least one marker gene for decidual senescent cells is DIO2; and / or The method of claim 1, wherein (c) the method comprises a step of detecting and / or quantifying the amount of PLA2G2A and DIO2.

3. a decreased level of said marker gene for decidual cells compared to a reference sample or level, and / or an increased level of the marker gene for decidual senescent cells compared to a reference sample or level; 10. The method of any one of the preceding claims, wherein the individual is indicated to be at risk of pregnancy loss or embryo implantation failure.

4. (a) detecting and / or quantifying in said biological sample the level of uterine natural killer (uNK) cells or the level of at least one marker gene for uNK cells, optionally wherein a decreased level of uNK cells or a decreased level of said at least one marker gene for uNK cells compared to a reference sample or level indicates that said individual is at risk of pregnancy loss or embryonic implantation failure; and / or (b) determining one or more risk indicators selected from the group consisting of maternal body mass index, maternal age, and number of previous pregnancy losses or embryo implantation failures; 10. The method of any one of the preceding claims, further comprising:

5. 1. A method for monitoring or evaluating the effectiveness of a treatment for reducing the risk of pregnancy loss or embryonic implantation failure in an individual, comprising the step of 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 monitoring or evaluating the effectiveness of the treatment, wherein the at least one marker gene for decidual cells comprises PLA2G2A.

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

7. (i) an increased level of the marker gene for decidual cells compared to a reference sample or level, and / or a decreased level of the marker gene for decidual senescent cells compared to a reference sample or level, indicates a positive response to the treatment; (ii) a decreased level of the marker gene for decidual cells compared to a reference sample or level, and / or an increased level of the marker gene for decidual senescent cells compared to a reference sample or level, indicates a negative response to treatment.

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

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

10. 1. A method for diagnosing a reproductive disorder in an individual, the method comprising the step of 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.

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

12. a decreased level of said decidual cell marker gene as compared to a reference sample or level, and / or an increased level of the decidual senescent cell marker gene as compared to a reference sample or level; 12. The method of claim 10 or 11, which indicates a positive diagnosis.

13. 13. The method of 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 decreased level of uNK cells or said at least one marker gene for uNK cells compared to a reference sample or level indicates a positive diagnosis.

14. In a positive diagnosis, the ratio of PLA2G2A to DIO2 is: (i) below the 50th percentile when compared to a reference sample or reference level; or (ii) below the 30th percentile when compared to a reference sample or reference level.

15. (a) the biological sample is an endometrial biopsy; and / or (b) The method of any one of the preceding claims, 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.

16. The individual, (a) suffers from or has suffered from infertility or embryo implantation failure after in vitro fertilization treatment; and / or (b) has already suffered at least one previous pregnancy loss or embryo implantation failure or is suffering from recurrent pregnancy loss.

17. (a) the method further comprises the step of detecting and / or quantifying genes that allow for the identification of days in the menstrual cycle, and optionally the genes that allow for the 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); and / or (b) The method of any one of the preceding claims, wherein the marker genes are detected and / or quantified using ELISA, Western blotting, immunohistochemistry, immunoassay, enzymatic 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. 18. A method of treating a reproductive disorder in an individual, comprising the steps of diagnosing the reproductive disorder according to the method of any one of claims 10 to 17, and administering to the individual who is positively diagnosed an agent or treatment regimen effective to treat the reproductive disorder.

19. (a) the agent or treatment regimen increases the level of decidual cells and / or uNK cells and / or decreases the level of decidual senescent cells in the individual; (b) the agent is a DPP4 inhibitor, and optionally 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 for decidual senescent cells is DIO2; and / or 20. The method of claim 18, wherein (e) the method comprises the step of administering progesterone and / or a progestogen.

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

21. 1. A method for selecting a patient for treatment to reduce the risk of embryo implantation failure or pregnancy loss, 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; 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 for decidual cells comprises PLA2G2A.

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

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

24. 10. A test kit suitable for use in the method of any one of the preceding claims, comprising means 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 means for detecting and / or quantifying in said individual the level of uNK cells or the level of at least one marker gene for uNK cells, wherein said at least one marker gene for decidual cells comprises PLA2G2A.

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