Biomarkers of pregnancy loss

Abstract

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

Description

[0001] BIOMARKERS OF PREGNANCY LOSS

[0002] Field of the Invention

[0003] The invention relates to improved methods for assessing the risk of pregnancy loss or embryo implantation failure, and also for monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure using specific biomarkers. The invention also relates to the use of these biomarkers in methods of diagnosing a reproductive disorder in an individual, and also to methods of treating a reproductive disorder. In addition, the biomarkers can further be used in methods of selecting patients for treatment to reduce risk of embryo implantation failure or miscarriage. The invention also relates to kits for use in any of the methods described herein.

[0004] Background to the Invention

[0005] Approximately 15% of clinical pregnancies result in pregnancy loss, most often during the first trimester (Rai & Regan, 2006). Two independent risk factors, maternal age and the number of previous pregnancy losses, have disproportionate effects on miscarriage rates (Magnus et al., 2019). The age-specific risk of miscarriage is accounted for by the increased incidence of meiotic errors in oocytes, driving the rapid increase in aneuploid embryos after the age of 35 years. On the other hand, the recurrence risk of miscarriage increases stepwise with approximately 10% with each additional miscarriage independently of maternal age (Magnus et al., 2019).

[0006] Implantation of the embryo requires intensive tissue remodeling of the endometrial stroma, driven by the postovulatory progesterone surge and rising intracellular cyclic adenosine monophosphate levels. This process, termed decidualization, is initiated during the midluteal phase of each cycle and involves differentiation of endometrial stromal cells (EnSC) into progesterone resistant (DIO2+) stromal cells, also known as decidual senescent cells, and progesterone responsive (PLA2G2A+) stromal cells, also known as decidual cells (as described in WO 2021 / 032973). PLA2G2A expression is induced in response to progesterone (in progesterone responsive cells). DIO2 expression is repressed in response to progesterone. Hence, progesteroneresponsive cells are PLA2G2A+ (PLA2G2A expression induced in response to induction by progesterone) and progesterone resistant cells are DIO2+ (DIO2 expression is not repressed in progesterone-resistant cells). PLA2G2A expression and DIO2 expression are therefore usually mutually exclusive, with responsiveness (characterized by PLA2G2A expression) and resistance (characterized by DIO2 expression) representing essentially binary states. Co-expression only occurs upon transition between progesterone resistant and responsive states. Accordingly, cells co-expressing both markers are rare.

[0007] The terms decidual senescent cells and progesterone resistant cells are used interchangeably herein. The terms decidual cells and progesterone responsive cells are also used interchangeably herein. Hence, references to decidual and decidual senescent cells can be construed as a reference to progesterone responsive cells, and progesterone resistant cells, respectively.

[0008] Upon progesterone-dependent differentiation (termed ‘decidualization’), PLA2G2A+stromal cells selectively secrete factors involved in uNK cells recruitment and activation regulating their proliferative expansion and differentiation into functionally and phenotypically distinct uterine NK (uNK) subsets. During the implantation window, there is a transition in the endometrium from DIO2+ stromal cells being dominant, to PLA2G2A+ cells becoming dominant. However, attempts to elucidate the link between uNK cells and pregnancy loss have produced contradictory results (Quack et al., 2001, Quenby et al., 1999, Clifford et al., 1999, Qu et al., 2008, Lachapelle et al., 1996, Yamamoto et al., 1999, Guo et al., 2021, Shimada et al., 2004, Michimata et al., 2002). The mechanism behind any proposed influence of uNK cells on miscarriage rates was previously unknown.

[0009] Despite the inconsistent findings, uNK cell testing is still practiced in the clinic, but the diagnostic pathway is complicated, labour intensive, and dependent on experienced staff. Moreover, inconsistent results may lead to women being mismanaged based on the test results (Moffett and Shreeve, 2015). Currently there are no diagnostic tests to (i) assess uNK cell subpopulations, (ii) evaluate the efficacy or therapeutic interventions prior to pregnancy, and (iii) inform patients and their doctors accurately of the risk of future losses of chromosomally normal pregnancies. The biomarkers described herein address the current gap based on bespoke endometrial diagnostics.

[0010] The inventors have discovered that balancing stromal decidual subpopulations at implantation is controlled by influx of uterine NK cells, with CD 160+ uNK3 cells targeting and eliminating stressed and damaged stromal cells, allowing expansion of progesterone responsive (which are referred to in Figure 2 as progesterone dependent, PLA2G2A+ cells) stromal cells (see Figure 2). Accordingly, biomarkers for these uNK cell subsets (e.g. ITGAD and CD 160) may be used as screening test that measures the risk of euploid (chromosomally normal) pregnancy loss. The discovery of the roles of uNK cell subpopulations and the utility of biomarkers for these enables further assessment of the endometrial ‘state’ during the midluteal implantation window before pregnancy and identify women at risk of subsequent miscarriage related to impaired uNK cell function. The use of biomarkers for uNK cell populations may be used to enhance the accuracy of existing tests, or as a standalone assessment.

[0011] Summary of the Invention

[0012] The inventors have discovered that during the menstrual cycle and before implantation in healthy pregnancies, uNK cells progress from cytotoxic uNK3 cells to cytokine producing uNKl cells, via uNK2 cells with an intermediate phenotype. An impaired transition from uNK3 cells to uNKl cells may result in pregnancy loss. The discovery of marker genes characteristic of these uNK cell subsets enables the identification of women at risk of pregnancy loss. Specifically, the present inventors have identified that Integrin Subunit Alpha D (ITGAD) is a marker characteristic of uNKl cells, and that CD 160 is a marker characteristic of uNK3 cells. uNK2 cells may also express ITGAD, but are typically CD39 negative, while uNKl cells are CD39 positive.

[0013] Use of biomarkers for uNKl, uNK2 and / or uNK3 cells can therefore identify patients with a specific immune dysfunction, namely, patients in whom the required uNK populations fail to expand within the endometrium during the menstrual cycle. Such a population of patients may include patients not identified as being at risk using existing tests of endometrial function, for example based on stromal biomarkers. For example, a patient who is identified as being at risk by the detection and / or quantification of the uNK subsets according to the invention may exhibit a normal balance of stromal cells, as detected using biomarkers for progesterone-responsive and resistant cells, for example.

[0014] More specifically, a low ratio of expression of progesterone-responsive to progesterone-resistant stromal cell biomarkers, exemplified by normalised PLA2G2A / DIO2 ratios < 25th percentile, identifies an aberrant endometrial state that is receptive for embryo implantation but also excessively prone to breaking down in pregnancy, thus causing bleeding and miscarriage. Even in patients with a normal balance of stromal cell subpopulations (exemplified by a normalised PLA2G2A / DIO2 ratio > 25th percentile), poor uNK cell expansion in isolation, or impaired transition from cytotoxic uNK3 cells to cytokine producing uNKl cells, exemplified by low normalised ITGAD / CD160 ratios (< 25th percentile), nonetheless predisposes for poor placenta formation without excessive risk of decidual breakdown in pregnancy. Accordingly, the test as described herein may identify a new patient population.

[0015] Clinically, these pregnancy losses may present as ‘missed’ or ‘silent’ miscarriages. Missed / silent miscarriages are diagnosed by ultrasound scanning and often involve a sequence of events, starting with early-onset fetal growth restriction, fetal bradycardia and then fetal demise in the absence of overt uterine bleeding. In other words, silent miscarriages may be distinguished from miscarriages which are not ‘silent’ by the absence of uterine bleeding or pelvic pain at the moment of fetal demise. Without medical intervention, the clinical manifestations of miscarriage, such as significant vaginal bleeding and pelvic pain, often follows days, if not weeks, after fetal demise. Accordingly, a means of predicting or diagnosing these ‘silent’ miscarriages is of huge potential clinical benefit.

[0016] The test as described herein may allow to newly identify individuals at risk compared to previous tests using stromal biomarkers. Addition of biomarkers for uNK cell subsets to an existing test for endometrial function can also improve the overall accuracy of the test. Moreover, patients with abnormalities in both the balance of stromal cell populations, and uNK cell subset populations, may represent individuals at even greater risk of miscarriage, and particularly recurrent miscarriage. Therefore, the use of uNK cell subsets as described herein can not only identify a clinically significant new population of individuals at risk of or who have undergone ‘silent’ miscarriage, but in combination with use of decidual and decidual senescent cell markers, may identify individuals most at risk, and who may be in greatest need of immediate treatment.

[0017] The test of the present invention is designed specifically to assess an endometrial cause of pregnancy loss prior to pregnancy and aid / direct therapeutic interventions. Due to its simplicity and ease, it has the potential to become a routine, quick and relatively inexpensive test for any women suffering pregnancy loss, even if for the first time. It is a simpler and less expensive alternative to the currently available commercial test aimed at determining the ‘window of implantation’ in IVF patients (described in EP2 333 107 Bl).

[0018] Thus, the present inventors have surprisingly identified that marker genes for uNKl cells, uNK2 cells and uNK3 cells can be used as biomarkers, alone or in combination, in methods for assessing the risk of pregnancy loss or embryo implantation failure, and also for monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure. The biomarkers described herein can also be used in methods of diagnosing a reproductive disorder in an individual, and also to assist methods of treating a reproductive disorder. In addition, these biomarkers can further be used in methods of selecting patients for treatment to reduce risk of embryo implantation failure or miscarriage, and methods of stratifying patients. The biomarkers may be detected using kits as described herein.

[0019] The present invention thus provides a method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uterine natural killer subset 3 (uNK3) cells, at least one marker gene for uterine natural killer subset 2 (uNK2) and / or at least one marker gene for uterine natural killer subset 1 (uNKl) cells, in a biological sample obtained from the individual, and thereby assessing the risk.

[0020] The present invention also provides a method of monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells, in a biological sample obtained from the individual and thereby monitoring or evaluating the effect of the treatment.

[0021] The present invention further provides a method of diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a sample obtained from an individual, thereby diagnosing the disorder.

[0022] The present invention also provides a method of treating a reproductive disorder in an individual, or of preventing pregnancy loss or embryo implantation failure in an individual, the method comprising diagnosing the reproductive disorder or assessing the risk of pregnancy loss or embryo implantation failure as described herein, and administering an agent or carrying out a treatment regimen effective to treat the reproductive disorder or prevent pregnancy loss or embryo implantation failure in the individual who is positively diagnosed or assessed as being at risk.

[0023] The invention also provides an agent for use in a method of treating a reproductive disorder in an individual, or of preventing pregnancy loss or embryo implantation failure in an individual, the method comprising diagnosing the reproductive disorder or assessing the risk of pregnancy loss or embryo implantation failure as described herein, and administering an agent or carrying out a treatment regimen effective to treat the reproductive disorder or prevent pregnancy loss or embryo implantation failure in the individual who is positively diagnosed or assessed as being at risk.

[0024] The present invention also provides a method of selecting patients for treatment to reduce risk of embryo implantation failure or pregnancy loss, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and selecting the patients for treatment to reduce the risk of pregnancy loss or embryo implantation failure based on the level of the marker genes.

[0025] The present invention also provides a test kit suitable for use in a method of any one of the preceding claims, wherein the test kit comprises means for detecting and / or quantifying at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker for uNKl cells in a biological sample obtained from an individual, optionally at a nucleic acid or protein level, and optionally further comprising means for detecting and / or quantifying the level of uNK cells in the individual.

[0026] The invention further provides a method of assessing readiness for conception or successful embryo implantation in an individual comprising detecting and / or quantifying at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker for uNKl cells in a biological sample obtained from an individual, and thereby assessing readiness for conception or successful embryo implantation.

[0027] The present invention provides a particularly advantageous endometrial test for recurrent pregnancy loss patients. The test is suitable for any woman planning pregnancy.

[0028] Brief Description of the Figures

[0029] Figure 1: Schematic showing the functionally distinct uNK cell subpopulations. It is proposed that uNK cells acquire sequentially killer cell immunoglobulin-like receptors (KIRs) and the ectonucleotidase CD39 as the menstrual cycle progresses, heralding the switch from a pro-inflammatory to a cytokine-producing phenotype (Strunz et al., 2021)

[0030] Figure 2: Schematic illustrating the proposed relationship between uNK3 cells and endometrial cells. Pre-decidual cells emerge as progesterone dependent (otherwise known as progesterone-responsive) PLA2G2A+ decidual cells upon closure of the implantation window, although some cells damaged by replication stress give rise to progesterone resistant decidual-like senescent cells. Clearance of damaged stromal cells is mediated by activated uNK3 cells, thereby preventing the emergence of decidual-like senescent cells responsible for tissue breakdown and menstruation.

[0031] Figure 3: uNK3 cells are involved in the clearance of progesterone resistant stromal cells. Endometrial stromal cells were decidualised and co-cultured with the three uNK cell subpopulations. Cellular senescence (as a proxy-marker of progesterone resistant cells) was monitored by senescence associated P galactosidase (SA- p -Gal) activity. Only uNK3 (KIR+CD39+) reduced SA-P-Gal activity significantly compared to control. Different letters above each treatment group indicates groups are significantly different from each other at P < 0.05 using the Friedman test and Dunn’s multiple comparison test. (n=10).

[0032] Figure 4: Single cell sequencing from 12 timed mid-luteal endometrial biopsies, a Left panel UMAP visualization of cells by broad cell type. Middle panel Visualization of all immune cells by cell type. Right panel Visualization of 4 uNK cell clusters: uNK-p (proliferative) cells, uNKl cells, uNK2 cells and uNK3 cells, b Violin and feature plots showing expression of the uNK cell specific biomarkers ITGAD and CD 160 unique to uNKl / 2 cells and uNK3 cells, respectively.

[0033] Figure 5: Proportion of uNK cell subpopulations in ES (early secretory), MS (mid secretory) and LS (late secretory) phases of the menstrual cycle. Note this shift from a uNK3 dominant state in the ES phase to a uNKl / 2 dominant state in the LS phase. (ES is defined as 2-5 days, MS as 6-10 days and LS 11-15 days post pre-ovulatory luteinizing hormone (LH) surge respectively.).

[0034] Figure 6: a Upper panel Expression of ITGAD and CD160 mRNA expression in proliferative and early-, mid-, and late-luteal phase endometrium. Each bar represents an individual biopsy. The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GEO Profiles ID: GDS2052). Lower panel ITGAD and CD160 mRNA levels quantified by ddPCR analysis in 853 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 for each day was 153 (range: 51-202). b Depiction of the menstrual cycle (standardized 28 day cycle used). The window of implantation coincides with the mid secretory phase of the cycle where a switch from a uNK3 CD160+ dominant state to a uNKl / 2 ITGAD+ dominant state is apparent.

[0035] Figure 7 a Dotplot indicating frequency of patients in each ITGAD / CD160 quartile on the basis of the number of previous pregnancy losses. Circle size reflects the percentage of patients, n numbers are indicated above each column, b Frequency of patients with ITGAD / CD160 ratio below the 25th percentile (left panel) and above the 75th percentile (right panel) Different letters above the graph indicate groups are significantly different to each other at Chi-squared test P < 0.05. c Correlations between ITGAD / CD160 percentile and serum hormone percentiles. Pearson’s r and significance are indicated, d Analysis of ITGAD / CD160 percentiles in endometrial biopsies obtained prior to a pregnancy that resulted in a live birth or miscarriage, e Top panel schematic of patient inclusion / exclusion on the basis of miscarriage karyotype. Bottom panel Odds ratio of subsequent miscarriage (M) or live birth (LB) in all pregnancy losses (left), or unknown and euploid pregnancy losses (right). Significant findings by Chi-square test indicated by dotted lines and open circles denotes P < 0.05. n.s.: non-significant (P > 0.05).

[0036] Detailed Description of the Invention

[0037] It is to be understood that different applications of the disclosed methods may be tailored to the specific needs in the art. It is also to be 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 supra or infra, are hereby incorporated by reference in their entirety.

[0038] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes “cells”, and the like.

[0039] Method for assessing the risk of pregnancy loss or embryo implantation

[0040] The invention provides a method for assessing the risk (or likelihood or probability) of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uterine natural killer subset 3 (uNK3) cells, at least one marker gene for uterine natural killer subset 2 (uNK2) and / or at least one marker gene for uterine natural killer subset 1 (uNKl) cells in a biological sample obtained from the individual, and thereby assessing the risk.

[0041] “Detecting” as referred to herein may comprise determining the presence of, or the absence of, a particular marker in a sample. Accordingly, detecting may comprise determining if a marker is expressed, or determining if a marker is not expressed. Detecting at least one marker gene may comprise or consist of detecting multiple marker genes, such as at least two, at least three, at least four, at least five, at least six or more marker genes.

[0042] In some embodiments, the method comprises detecting and / or quantifying at least one marker gene for uNKl cells. In a preferred embodiment, the at least one marker gene for uNKl cells comprises or consists of Integrin Subunit Alpha D (ITGAD). In some embodiments, the at least one marker gene for uNKl cells comprises or consists of at least one killer immunoglobulin-like receptor (KIR) and / or CD39. In some embodiments, the at least one marker gene for uNKl cells comprises or consists of ITGAD, KIR and / or CD39.

[0043] In some embodiments, the method comprises detecting and / or quantifying at least one marker gene for uNK3 cells. In a preferred embodiment, the at least one marker gene for uNK3 cells comprises or consists of CD 160.

[0044] In some embodiments, the method comprises detecting and / or quantifying at least one marker gene for uNK2 cells. In a preferred embodiment, the at least one marker gene for uNK2 cells comprises or consists of Integrin Subunit Alpha D (ITGAD). In some embodiments, the at least one marker gene for uNK2 cells comprises or consists of at least one killer immunoglobulin-like receptor (KIR). In some embodiments, the at least one KIR may be KIR2DL1 or KIR2DL4. In some embodiments, the detecting of at least one marker gene for uNK2 cells comprises or consists of detecting ITGAD and detecting the absence of CD39 expression.

[0045] In a preferred embodiment, the method comprises detecting and / or quantifying at least one marker gene for uNKl cells and at least one marker gene for uNK3 cells. In a preferred embodiment, the method comprises detecting ITGAD and CD160.

[0046] Any further marker genes for uNKl, uNK2 and / or uNK3 cells may be selected from any such marker genes. Thus, the marker genes may be any genes characteristic of uNKl, uNK2 or uNK3 cells and which are detectible in the biological sample, such as an endometrial sample. For example, a marker gene for uNKl cells may be any marker whose decrease is indicative of a decreased level or number of uNKl cells, and a marker gene for uNK3 cells may be any marker whose increase is indicative of an increased level of uNK3 cells.

[0047] The level of the marker genes is typically compared with a control sample or reference sample or level. Any suitable control sample or reference sample or level may be used. A control sample or reference sample or level may represent a normal or healthy sample / level, for example obtained or determined from an individual or multiple individuals not having any reproductive disorder, or not having had pregnancy loss or embryo implantation failure. The individual(s) may have had one or more successful pregnancies. The control or reference sample or level may also be obtained or determined from an individual or multiple individuals who have responded positively to treatment to reduce the risk of pregnancy loss or embryo implantation failure. Alternatively, a control sample or reference sample or level may represent a sample / level from an individual or multiple individuals having a reproductive disorder, or who have had one or more miscarriages or embryo implantation failures, 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 may be obtained or the results may 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 that is obtained on or around the same day of the menstrual cycle as the test sample. Other suitable control or reference samples or levels can readily be identified by the person skilled in the art.

[0048] A decreasing or decreased level of a marker gene for uNKl cells e.g. ITGAD in a sample, as compared with a reference sample or level, may indicate that the individual is at risk of pregnancy loss or embryo implantation failure. Alternatively, an increasing or increased level of a marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, may indicate that the individual is at risk of pregnancy loss or embryo implantation failure. The level of marker gene can be determined using any methods described herein and that are known to the skilled person. In one instance, a decreasing or decreased level of a marker gene for uNKl cells e.g. ITGAD, as compared with a reference sample or level, and an increasing or increased level of a marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, together indicate that the individual is at risk of pregnancy loss or embryo implantation failure.

[0049] In one instance, the method for assessing the risk of pregnancy loss or embryo implantation failure in an individual further comprises detecting and / or quantifying the level of uterine natural killer (uNK) cells in the sample, for example based on the level of at least one marker gene common to all uNK cell subsets in the sample.

[0050] A decreasing or decreased level of the uNK cells or uNK cell gene markers in a sample, as compared with a reference sample or level, may indicate that the individual is at risk of pregnancy loss or embryo implantation failure.

[0051] In some embodiments, the method comprises detecting and / or quantifying the amount of uterine natural killer subset 3 (uNK3) cells, uterine natural killer subset 2 (uNK2) and / or uterine natural killer subset 1 (uNKl) cells in a biological sample obtained from the individual, and thereby assessing the risk. In some embodiments, the marker gene may be detected and / or quantified by using a labelled antibody to detect the marker gene in its protein form. Exemplary techniques using labelled antibodies may include flow cytometry. In some embodiments, the detecting and / or quantifying may be performed without reference to marker genes.

[0052] The method of the invention may also further comprise detecting and / or quantifying genes that allow identification of the day in the menstrual cycle as described below.

[0053] Further risk indicia, which may be used to assess the risk of pregnancy loss or embryo implantation failure, include maternal body mass index (BMI), maternal age, number of previous pregnancy losses or embryo implantation failures, familial and intergenerational factors, history of infertility, placental abnormalities, cervical and uterine anomalies, smoking, alcohol consumption, etc. Additional risk indicia are known to those of skill in the art.

[0054] For instance, 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 maternal age is 35 and above, if there has been a history of recurrent pregnancy loss (e.g. when a woman has had 2 or more pregnancy losses before the pregnancies reached 20 weeks).

[0055] Samples

[0056] As used herein, the term “biological sample” or “sample” refers to any sample that is taken from an individual. Suitable samples in the context of the methods of the present invention include, for example, endometrial tissue, endometrial secretions, cells obtained from the endometrium, or an endometrial biopsy sample.

[0057] A sample obtained from the endometrium may be collected by any method known in the art, including through an endometrial biopsy or endometrial sampling. The technique involves removing a piece of tissue from the inner lining of the uterus (endometrium). The sample may also be obtained using a dilation and curettage procedure.

[0058] The sample may be or may 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 be or have been stored or preserved, e.g. by freezing, prior to use.

[0059] The sample may be taken during the luteal phase of the menstrual cycle. The luteal phase begins with the formation of the corpus luteum, with progesterone being significantly higher than in other phases of the menstrual cycle. The sample may be taken during the mid-luteal phase of the menstrual cycle. The sample is thus typically taken post-ovulation. The sample is typically taken during the embryo implantation window (also known as the receptivity window), of the menstrual cycle, in which the endometrium is receptive to implantation of an embyro. The embryo implantation window may be determined by any means, and may for example be calculated based on an ovulation test. An ovulation test may be based on hormone level, such as luteinizing hormone (LH) level (for example LH level in urine) or oestrogen level (for example based on salivary ferning). Alternatively, the level of one or more markers indicative of receptivity to embryo implantation may be determined. The sample may be taken between about 5 and about 11 days after an increase or surge (such as a 2-5 fold increase or surge) in the level of LH s, i.e. LH+5 to LH+11. LH is produced by the pituitary gland and is generally secreted at very low levels throughout the menstrual cycle, with the ovulatory phase of the menstrual cycle however beginning with a surge in LH.

[0060] The sample type and timing of sampling described above is applicable to any of the methods of the present invention detecting marker levels.

[0061] Individual

[0062] The individual referred to in any of the methods of the invention may be a human or a non-human menstruating mammal. The methods described herein may thus be applied in a veterinary context. The subject is preferably a human female.

[0063] The individual may be suffering or have suffered from infertility or embryo implantation failure. For instance, the individual may suffer from or have suffered from embryo implantation failure following in vitro fertilisation treatment. The individual may have suffered from at least one previous pregnancy loss or multiple pregnancy losses, and / or at least one embryo implantation failure or multiple embryo implantation failures. The individual may suffer from recurrent pregnancy loss (RPL). The individual may already be considered to be at risk of pregnancy loss or embryo implantation failure. The individual may be considered at risk of pregnancy loss or embryo implantation failure due to the presence of one or more of the risk indicia, including low or high body mass index (BMI), maternal age, number of previous pregnancy losses or embryo implantation failures, familial and intergenerational factors, history of infertility, placental abnormalities, cervical and uterine anomalies etc. Additional risk indicia for pregnancy loss or embryo implantation failure are known to those of skill in the art.

[0064] Pregnancy loss

[0065] Pregnancy loss refers to the failure of an embryo to result in a baby, which may result from unsuccessful embryo implantation. Pregnancy loss as assessed according to the biomarkers of the present invention is typically loss in the first trimester, particularly in the first 20 to 23 weeks of gestation, which may also be referred to as “miscarriage”. Typical symptoms of a miscarriage comprise vaginal bleeding with or without pain, and also cramping and pain in the lower abdomen. When at least two or more miscarriages occur, an individual may be diagnosed as suffering from recurrent pregnancy loss (RPL) or infertility. However, RPL may not be a binary state and there may be an increased risk of further pregnancy loss with any previous loss. The invention allows for the detection of any predisposition to RPL irrespective of the number of previous losses.

[0066] A 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 an embryo.

[0067] In a method of the present invention, the risk of pregnancy loss / miscarriage may be assessed. The method of the invention is preferably used for determining the risk of euploid miscarriage, and where the cause of pregnancy loss is not due to a chromosomal abnormality or error in an embryo. The method of the invention is preferably used for determining the risk of recurrent pregnancy loss / miscarriage. When determining or assessing the risk of recurrent miscarriage, it is preferable to detect and / or quantify the level of marker genes for uNK cell subsets (preferably uNKl and uNK3 subsets) and also for progesterone-responsive (decidual) stromal cells and progesterone-resistant (decidual senescent) stromal cells. Accordingly, in preferred embodiments, the method comprises detecting and / or quantifying the level of at least one marker gene for uNKl cells, at least one marker gene for uNK3 cells, at least one marker gene for progesterone-responsive (decidual) stromal cells and at least one marker gene for progesterone-resistant (decidual senescent) stromal cells. In such embodiments, a ratio of the level of the marker gene for uNKl cells to the level of the marker gene for uNK3 cells which is decreased relative to a reference sample, combined with a ratio of the level of the marker gene for progesteroneresponsive (decidual) stromal cells to the level of the marker gene for progesterone- resistant (decidual senescent) stromal cells which is decreased relative to a reference sample, indicates that an individual is at risk of, or is suffering from, recurrent miscarriage (which may also be termed ‘higher order miscarriage’).

[0068] In preferred embodiments, the miscarriage is silent miscarriage. Silent miscarriage is a miscarriage in which fetal demise is not accompanied by simultaneous clinical manifestation of pelvic pain or uterine bleeding. In embodiments of the methods described herein, a ratio of the level of the marker gene for uNKl cells to the level of the marker gene for uNK3 cells which is decreased relative to a reference sample, combined with a ratio of the level of the marker gene for progesterone-responsive (decidual) stromal cells to the level of the marker gene for progesterone-resistant (decidual senescent) stromal cells which unchanged relative to a reference sample, indicates that an individual is at risk of, or has undergone, silent miscarriage.

[0069] Embryo implantation failure

[0070] After fertilisation, a fertilised egg (or zygote) begins to divide by mitosis to produce an embryo. The process of the embryo attaching to the lining of the uterus, i.e. the endometrium, is known as implantation. “Embryo implantation failure” or “implantation failure” in accordance with the invention refers to failure of an embryo to be implanted into the endometrium. Implantation failure may occur either where patients are trying to conceive naturally, without any fertility treatment or after undergoing assisted reproductive technology e.g. in vitro fertilisation (IVF).

[0071] Some cases of implantation failure are thought to be caused by chromosomal abnormalities in the embryo, such as aneuploidy, e.g. autosomal trisomy, monosomy X, triploidy, tetraploidy etc. Other cases of embryo implantation failure are not due to chromosomal abnormalities or errors in an embryo. Some cases of implantation failure related to IVF are caused by poor embryo quality, the age of the eggs, a lack of response to IVF medication or other lifestyle factors (e.g. smoking).

[0072] According to the present invention, the risk of any case of embryo implantation failure may be assessed in any setting. The method of assessing risk of embryo implantation failure of the invention may thus be used for determining risk of implantation failure following natural conception, or the risk of implantation failure following assisted reproduction in an individual, for example following in vitro fertilisation. The method is preferably used for determining the risk of embryo implantation failure that is not due to a chromosomal abnormality or error in an embryo. The method may preferably be used to determine the risk of recurrent embryo implantation failure. uNK cell subsets and marker genes

[0073] In accordance with the method of assessing risk of pregnancy loss or implantation failure of the present invention, at least one marker gene for uterine natural killer subset 3 (uNK3) cells, at least one marker gene for uterine natural killer subset 2 (uNK2) and / or at least one marker gene for uterine natural killer subset 1 (uNKl) cells are detected and / or quantified.

[0074] During decidualization, the cells of the endometrium undergo significant changes in preparation for and during pregnancy. During this process, endometrial stromal cells (EnSC) either become a specialised cell (i.e. decidual cell) or become acutely senescent (i.e. decidual SNC). The inventors have found that specific uNK cell subsets have distinct roles in regulating the balance between decidual and decidual senescent cells. uNK3 (CD160+) cells are involved in the clearance of progesterone resistant stromal cells (Figure 3), whereas uNKl / 2 cells expand as the cycle progresses and confer immune tolerance to the implanting embryo and invading placental cells as well as promote angiogenesis and vascular remodelling (Strunz et al., 2021, Moffett and Shreeve, 2022). The inventors have found that an impaired transition from uNK3 to uNKl before the implantation window is associated with increased risk of pregnancy loss.

[0075] In accordance with the method of assessing risk of pregnancy loss or implantation failure of the present invention, at least one marker gene for uterine natural killer subset 3 (uNK3) cells, at least one marker gene for uterine natural killer subset 2 (uNK2) and / or at least one marker gene for uterine natural killer subset 1 (uNKl) cells may be detected. In some embodiments, the at least one marker gene is additionally quantified. The at least one marker gene for uNK3 cells may comprise CD 160, the at least one marker gene for uNK2 cells may comprise a killer immunoglobulin receptor (KIR) and / or ITGAD, and the at least one marker gene for uNKl cells may be selected from ITGAD, a KIR and CD39. The KIR may be KIR2DL1 or KIR2DL4 for example. The method may comprise detecting decreased expression of ITGAD, KIR and / or CD39. In preferred embodiments, the method comprises quantifying the amount of, or the expression of, CD 160. The method may comprise detecting increased expression of CD 160.

[0076] Preferably, the at least one marker gene for uNKl cells is ITGAD. Preferably, the at least one marker gene for uNK3 cells is CD 160. In a preferred embodiment, the method comprises detecting and / or quantifying the amount of both CD160 and ITGAD. The method may comprise detecting decreased expression of ITGAD and increased expression of CD 160, and thereby determining that an individual is at risk of pregnancy loss and / or embryo implantation failure. Any of the above genes may be detected and / or quantified in the method for assessing the risk of pregnancy loss or embryo implantation failure of the invention, as well as in further methods described below.

[0077] Centile or percentile graphs may be employed to compare expression levels of marker genes such as marker genes of uNKl, uNK2 or uNK3 cells (and also levels of other markers as discussed below) in samples obtained at different days in the menstrual cycle. Centile graphs are based on the statistical distribution of the expression levels of a given marker gene on a given day in the menstrual cycle, e.g. following a positive ovulation test. The more samples used to generate the centile graphs, the more accurate the reference range. For instance, centile graphs 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 of a given marker gene (i.e. centile) in a sample of an individual obtained on a given day in the menstrual cycle may be calculated against the reference percentile graph.

[0078] In some instances, the determination of the centile for each marker gene allows the cause and clinical presentation of the individual to be determined, such as that for recurrent pregnancy loss. Putative defects, or decidual dyshomeostasis, along the decidual pathway may be determined. In one example, a low level of a uNKl cell marker gene and a high level of a uNK3 cell marker gene, as compared with a reference sample or reference level, occurs more frequently in recurrent pregnancy loss. In a particular instance, where the ratio of ITGAD to CD 160 is below the 50thpercentile as compared with a reference sample or reference level, then there is a positive diagnosis. In other instances, where the ratio of ITGAD to CD160 is below the 40thpercentile, the 30thpercentile, the 20thpercentile, or the 10thpercentile, as compared with a reference sample or reference level, then there is a positive diagnosis. In preferred embodiments, where the ratio of ITGAD to CD 160 is below the 25thpercentile, there is a positive diagnosis.

[0079] In other instances, the invention may comprise determining the relative risk of pregnancy loss such as miscarriage. For example, an individual may be found to be twice as likely to miscarry as to have a live birth. In other aspects, the individual may be found to be three, four or five times as likely to miscarry. In one particular instance, an individual may be found to be twice as likely to miscarry when the ratio of ITGAD to CD 160 is below the 25thpercentile. uNK cells and uNK cell gene markers

[0080] In accordance with the method of assessing risk of pregnancy loss or implantation failure of the invention, in addition to the marker genes for the specific uNK cell subsets, the numbers and / or amounts of uNK cells, and the expression of marker genes thereof, may also be detected and / or quantified, typically by detection of one or more marker genes for uNK cells in general.

[0081] The level of uNK cells in the sample may be detected and / or quantified by any means known in the art. uNK cells may be detected and / or quantified using immunohistochemistry and image analysis. Alternatively, uNK cells may be detected based on the level of at least one marker gene for uNK cells in the sample.

[0082] In accordance with the methods of the present invention, any uNK cell gene marker may be detected and / or quantified. Marker genes for uNK cells may be selected from NCAM1, KLRB1, KLRC1, GZMA, GZMB, IL2RB and IL2RG. The above genes may be detected and / or quantified in the method for assessing the risk of pregnancy loss or embryo implantation failure described herein, and also in further methods of the invention detecting marker genes as described below. uNK cell deficiency occurs more frequently in recurrent pregnancy loss. Decidual cells and decidual senescent cells and marker genes

[0083] Successful transition of the endometrium (from a cycling tissue into a semipermanent tissue capable of maintaining the placenta throughout pregnancy) is not only dependent on the transition of the uNK cell subsets from uNK3 to uNKl. Endometrial remodelling is also dependent on stress-resistant decidual cells co-opting uterine natural killer (uNK) cells to eliminate their acutely stressed counterparts, i.e. decidual senescent cells, through granule exocytosis. Thus, the balance of diverging decidual populations and uNK cells during the midluteal phase of the menstrual cycle is described herein to likely determine the ability of the endometrium to transition into a pregnancy tissue. Imbalance in decidual subsets is also linked herein to reproductive failure. Thus, determination of the level of uNK cells in combination with detecting levels of markers for decidual and decidual senescent cells provides additional information for assessment of the risk of pregnancy loss or implantation failure and also in relation to diagnosis of reproductive disorders more generally.

[0084] In accordance with the method of assessing risk of pregnancy loss or implantation failure of the present invention, the method may further comprise detecting and / or quantifying the amount of at least one marker for decidual cells, and / or a least one marker for decidual senescent cells.

[0085] During decidualization, the cells of the endometrium undergo significant changes in preparation for and during pregnancy. During this process, endometrial stromal cells (EnSC) either become a specialised cell (i.e. decidual cell) or become acutely senescent (i.e. decidual SNC).

[0086] The decidual cell detected may be any decidual cell and the decidual senescent cell detected any decidual senescent cell, typically any such cells in an endometrial sample. A decidual or decidual senescent cell is typically derived from an endometrial stromal cell. Decidual cells are stress-resistant and are also described herein as stress-resistant decidual cells.

[0087] In accordance with the method of assessing risk of pregnancy loss or implantation failure of the present invention, at the method may further comprise detecting and / or quantifying the amount of at least one marker for decidual cells, and / or a least one marker for decidual senescent cells. The at least one marker gene for decidual cells may comprise one or more of PLA2G2A, SCARA5, FTL, GLRX and IL1RL1, and the marker genes for decidual senescent cells typically comprise one or more DIO2, CLU and IGFBP1.

[0088] Preferably, the at least one marker gene for decidual cells is PLA2G2A. The method may comprise detecting a decrease in PLA2G2A. Preferably, the at least one marker gene for decidual senescent cells is DIO2. The method may comprise detecting an increase in DIO2. In a preferred embodiment, the at least one marker gene for decidual senescent cells is DIO2, and the method comprises detecting and / or quantifying the amount of both PLA2G2A and DIO2. The method may comprise detecting a decrease in PLA2G2A and an increase in DIO2.

[0089] Any of the above genes may be detected and / or quantified in the method for assessing the risk of pregnancy loss or embryo implantation failure of the invention, as well as in further methods described below.

[0090] In some embodiments, the amount of the at least marker gene for decidual cells and / or the amount of the at least one marker gene for decidual senescent cells does not differ or does not significantly differ from the amount of the marker gene in a reference sample, such as a reference sample taken from an individual or individuals who do not have a reproductive disorder. In other words, the detecting and / or quantifying of the amount of at least one marker for decidual cells, and / or a least one marker for decidual senescent cells indicates that the individual has a normal balance of stromal cells. Nonetheless, in such embodiments, the detection and / or quantification of marker genes for uNK cell subsets may identify individuals at risk of pregnancy loss or implantation failure. Thus, in some embodiments of any of the methods of the invention, the individual has a normal balance of stromal cells. In some embodiments, the individual has an amount of the marker gene for decidual cells and / or the amount of the marker gene for decidual senescent cells which does not differ from the amount of the marker gene in a reference sample taken from an individual or from a group of individuals without a reproductive disorder. By “does not differ”, it is meant typically the amount of the marker genes does not differ in a statistically significant manner between the reference sample (taken from individual(s) without a reproductive disorder) and test sample (taken from individual to be tested). Appropriate statistical tests may be identified and performed by the skilled person. In some embodiments, the ratio of the amount of the marker gene for decidual cells to the amount of the marker gene for decidual senescent cells is measured. In some embodiments, the ratio of the amount of the marker gene for decidual cells to the amount of the marker gene for decidual senescent cells does not differ from the ratio determined for a reference sample. In some embodiments, the ratio of the amount of the marker gene for decidual cells to the amount of the marker gene for decidual senescent cells differs from the ratio determined for a reference sample. In some embodiments the individual has an amount of the marker gene for decidual cells and / or an amount of the marker gene for decidual senescent cells which differs from that of a reference sample, and the individual also has a balance of uNK cell subsets (as quantified by e.g. the ratio of the amount of a marker gene for uNKl cells to the amount of a marker gene for uNK3 cells) which differs from that of a reference sample. Such an individual may be at particular risk of pregnancy loss such as miscarriage or implantation failure.

[0091] Centile or percentile graphs may be employed to compare expression levels of marker genes such as marker genes of decidual and decidual senescent cells (and also levels of other markers as discussed below) in samples obtained at different days in the menstrual cycle. Centile graphs are based on the statistical distribution of the expression levels of a given marker gene on a given day in the menstrual cycle, e.g. following a positive ovulation test. The more samples used to generate the centile graphs, the more accurate the reference range. For instance, centile graphs 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 of a given marker gene (i.e. centile) in a sample of an individual obtained on a given day in the menstrual cycle may be calculated against the reference percentile graph.

[0092] In some instances, the determination of the centile for each marker gene allows the cause and clinical presentation of the individual to be determined, such as that for recurrent pregnancy loss. Putative defects, or decidual dyshomeostasis, along the decidual pathway may be determined. In one example, a low level of a decidual cell marker gene e.g. PLA2G2A and a high level of decidual senescent cell marker gene e.g. DIO2, as compared with a reference sample or reference level, is indicative of excessive decidual senescence, and occurs more frequently in recurrent pregnancy loss. In a particular instance, where the ratio of PLA2G2A to DIO2 is below the 50thpercentile as compared with a reference sample or reference level, then there is a positive diagnosis. In other instances, where the ratio of PLA2G2A to DIO2 is below the 40thpercentile, the 30thpercentile, the 20th percentile, or the 10thpercentile, as compared with a reference sample or reference level, then there is a positive diagnosis.

[0093] In other instances, the invention may comprise determining the relative risk of pregnancy loss or specifically miscarriage. For example, an individual may be found to be twice as likely to miscarry as to have a live birth. In other aspects, the individual may be found to be three, four or five times as likely to miscarry. In one particular instance, an individual may be found to be twice as likely to miscarry when the ratio of PLA2G2A to DIO2 is below the 15thpercentile.

[0094] Determination of the timing in the menstrual cycle

[0095] In accordance with the methods of the present invention, a determination of the time (such as the point, stage or day) in the menstrual cycle on which the sample is obtained may be additionally carried out. Any parameter including any known hormone, marker or other parameter (including any hormone or marker described above) that allows timing of the point, stage or day in the cycle may be used. Preferably, the sample is obtained in the embryo implantation window, and the point, stage or day within the embryo implantation window is determined. The determination of the point, stage or day in the menstrual cycle advantageously allows a sample to be compared to a reference sample or level representative of the same point, stage or day, since the hormone levels or marker gene levels change throughout the cycle.

[0096] In a particular embodiment, in addition to marker genes for uNK cell subsets, decidual cells, decidual senescent cells and / or uNK cells in general, marker genes that allow identification of the point, stage or day in the menstrual cycle are thus also detected and / or quantified. Such marker genes are also referred to herein as molecular timing genes, and are typically indicative of timing in the implantation window. Through the analysis of these genes, the accuracy of the detection based on analysis of the marker genes for uNK cell subsets, decidual cells, decidual senescent cells and / or uNK cells in general may be improved.

[0097] The purpose of molecular timing is two-fold. Because of cycle-dependence of gene marker levels, the interpretation of the levels of the decidual cell gene markers, the decidual senescent cell gene markers, and / or uNK cell levels or uNK cell subset gene markers, is advantageously assisted by knowledge of the day in the cycle a biopsy is taken. Practically, this may also be achieved by scheduling the biopsy relative to the preovulatory luteinising hormone (LH) surge as discussed above. The methods of the invention may thus be carried out in an individual by obtaining a sample at a suitable time point subsequent to an LH surge as described above. However, by considering molecular timing based on analysis of marker gene expression, the risk of erroneous timing of the biopsy due to patient error and intrinsic variation between the LH surge and the exact time of ovulation may be reduced.

[0098] The window of implantation (also known as the receptivity window) is associated with dramatic changes in gene expression in the glandular epithelium. Thus, the method of the invention may comprise detection of any marker gene having a change in expression (and which is typically selectively expressed) in the glandular epithelium during the implantation window and thus able to report on the point, stage or day in the embryo implantation window. The determination may be based on two or more genes that are selectively expressed in the glands and that exhibit opposing expression profiles as the menstrual cycle progresses. The ratio of two or more such genes may be determined.

[0099] Marker genes that enable determination of the molecular timing of the embryo implantation window and may be used according to the invention include any one or more of GPX3, DPP4 (a GPX3-like gene), SLC15A2 and CTNNA2 (a SLC15A2-YkQ gene). Preferably, the genes that allow identification of the timing of the day in the menstrual cycle may comprise, consist of, or consist essentially of, GPX3 and SLC15A2. The ratio of GPX3 and SLC15A2 may thus be determined. Because molecular timing as used in the invention is typically based on genes selectively expressed in epithelial cells such as GPX3 and SC15A2, whereas decidual cell and decidual senescent cell markers e.g. PLA2G2A and DIO2 are selective stromal cell markers, molecular timing may also be used to diagnose asynchrony between the hormonal response in the epithelial and stromal compartment. As described herein, GPX3 and SLC15A2 are regulated in opposing ways as the luteal phase unfolds (i.e. GPX3 is rapidly upregulated whereas SLC15A2 is rapidly downregulated). Consequently, the ratio of these two genes changes profoundly from day to day during the implantation window. The ratio between GPX3 and SLC15A2 rises markedly across LH+5 and LH+11 days of the cycle and thus the ratio between GPX3 and SLC15A2 may be matched to a particular day in the cycle. The particular GPX3 / SLC15A2 ratio may be matched to a particular day in the cycle based on a centile / percentile graph plotted from values obtained from pooled reference samples for that particular day in the cycle, for example as obtained by detection of the LH surge (e.g. using home ovulation kits). Preferably, if the GPX3 / SLC15A2 ratio in a test sample falls between the 25thto 75thpercentile of a reference centile / percentile graph plotted from values obtained from pooled reference samples for a particular day in the cycle, and matches (i.e. is congruent) with the day of the luteal phase as determined by detection of the LH surge (e.g. using a home ovulation kit), then the timing of a biopsy may be considered improved in accuracy, and test results may be reported on the basis of the day in the cycle as determined by detection of the LH surge e.g. using a home ovulation kit. If the GPX3 / SLC15A2 ratio in a test sample falls outside of the 25thto 75thpercentile of a reference centile / percentile graph plotted from values obtained from pooled reference samples for a particular day in the cycle (i.e. is incongruent), or if the GPX3 / SLC15A2 ratio does not match with the day of the luteal phase as determined by detection of the LH surge (e.g. using a home ovulation kit), then the timing of a biopsy may be considered less accurate, and test results may be reported both on the basis of the day in the cycle as determined by detection of the LH surge e.g. using a home ovulation kit, as well as based on molecular timing results

[0100] In preferred embodiments, any method of the invention may comprise testing and / or quantifying the amount of ITGAD and CD 160, PLA2G2A and DIO2, and GPX3 and SLC15A2.

[0101] Marker gene sequences

[0102] Particular sequences for marker genes useful in accordance with the present invention, with their database accession / identification number in the NCBI Gene database, Ensembl database and OMIM database are disclosed herein. The gene sequences as disclosed herein include those available with reference to these online sequence databases as of 16 June 2019. Thus, below is a list of marker genes with representative accession numbers (in parentheses: NCBI Gene database, followed by Ensembl database, followed by OMIM database) and alternative gene names (in italics):

[0103] Phospholipase A2 Group IIA: PLA2G2A (5320, ENSG00000188257, 172411) M0M1, PLA2, PLA2B, PLA2L, PLA2S, PLAS1, sPLA2

[0104] Scavenger Receptor Class A Member 5: SCARA5 (286133, ENSG00000168079, 611306) Tesr, NET33, FLJ23907, MGC45780 Ferritin Light Chain: FTL (2512, ENSG00000087086, 134790) LFTD, NBIA3, MGC71996;

[0105] Glutaredoxin: GLRX(2745, ENSG00000173221, 600443) GRX, GRX1;

[0106] Interleukin 1 Receptor Like V. IL1RL1 (9173, ENSG00000115602, 601203) Tl, ST2, DER4, ST2L, ST2V, FIT-1, IL33R; lodothyronine Deiodinase 2: DIO2 (1734, ENSG00000211448, 601413) D2, 5DII,

[0107] SelY, DIOII, TXDI2,-

[0108] Clusterin: CLt / (1191, ENSG00000120885, 185430) CLI, AAG4, APOJ, CLU1,

[0109] CLU2, KUB1, SGP2, APO-J, SGP-2, SP-40, TRPM2, TRPM-2, NA1 / NA2,-

[0110] Insulin Like Growth Factor Binding Protein 1 : IGFBP1 (3484, ENSG00000146678, 146730) AFBP, IBP1, PPI 2, IGF-BP25, hIGFBP-1,

[0111] Glutathione Peroxidase 3: GPX3 (2878, ENSG00000211445, 138321) GPx-P, GSHPx-3, GSHPx-P;

[0112] Solute Carrier Family 15 Member 2:

[0113] SLC15A2(6565, ENSG00000163406, 602339) PEPT2;

[0114] Dipeptidyl-peptidase IV: DPP4 (1803, ENSG00000197635, 102720) CD26,

[0115] ADABP, ADCP2, DPPIV, TP103;

[0116] Catenin Alpha 2: CTNNA2 (1496, ENSG00000066032, 114025) CAPR, CTNR,

[0117] CAP-R, CT114, CDCBM9;

[0118] Interleukin 2 Receptor Subunit Beta: IL2RB

[0119] (3560, ENSG00000100385, 146710) CD122, IL15RB, P70-75

[0120] Interleukin 2 Receptor Subunit Gamma: IL2RG

[0121] (3561, ENSG00000147168, 308380) P64, CIDX, IMD4, CD132, SCIDX, IL-2RG,

[0122] SCIDX1;

[0123] Neural Cell Adhesion Molecule 1 : NCAM1

[0124] (4684, ENSG00000149294, 116930) CD56, NCAM,MSK39.

[0125] Integrin subunit alpha D: ITGAD

[0126] (3681, ENSG00000156886, 602453) AB£>2, GDI ID;

[0127] CD 160 molecule: CD 160

[0128] (11126, ENSG00000117281, 604464) NK1, BY55, NK28;

[0129] CD38 molecule: CD38

[0130] (952, ENSG00000004468, 107270) ADPRC1, cADPRl, ADPRC 1; Killer cell immunoglobulin like receptor, two 1g domains and long cytoplasmic tail

[0131] 4 : KIR2DL4

[0132] (3805, ENSG00000189013, 604945) G9P, CD158D, KIR103, KIR-2DL4, KIR103AS, KIR-103AS;

[0133] Killer cell immunoglobulin like receptor, two 1g domains and long cytoplasmic tail . KIR2DL1

[0134] (3802, ENsG00000125498, 604936) NKAT, NKATl, p58, CD158A, KIR221, NKAT-1, KIR-K64, KIR2DL3

[0135] Detection and / or quantification of the amount / level of the biomarkers

[0136] As used herein, the term “marker gene” or “biomarker” refers to a gene, or a fragment of a gene, the change in amount and / or the detection of which can be correlated with a particular physical condition or state. Particular marker genes used in the present invention are correlated with the risk of pregnancy loss or embryo implantation failure, and are also used in the methods described herein. The detection and / or quantification of such marker genes may be achieved by any means and is not limited to detection / quantification of nucleic acids. Marker genes may also be detected via their respective expression products, including the expressed peptides, polypeptides, and proteins, and fragments thereof. In some embodiments, the marker gene may be detected and / or quantified by using a labelled antibody to detect the marker gene in its protein form. Exemplary techniques using labelled antibodies may include flow cytometry.

[0137] As used herein, the term “amount” or “level” as used herein refers to a quantity of a marker gene or its expression product that is detectable or measurable in a biological sample and / or control or reference sample. The quantity of a marker gene can be, for example, a quantity of nucleic acid or protein. The term can alternatively include combinations thereof. The amount or level of the marker genes may refer to the absolute amount or level of the biomarkers. Alternatively, a change in the relative level or amount of marker(s) may be assessed by comparing the level or amount of the marker genes in a sample from the subject with a control value or reference value. Alternatively, the relative amount or level of the marker genes may in some instances refer to the concentration of the marker genes relative to the total amount or level of marker genes in the sample. The level of marker genes can be detected and / or quantified by detection of nucleic acid, e.g. RNA. For example, levels of mRNA can be measured by reverse transcription quantitative polymerase chain reaction (RT- PCR followed with qPCR). RT-PCR is used to create a cDNA from the mRNA. The cDNA can be used in a qPCR assay to produce fluorescence as the DNA amplification process progresses. By comparison to a standard curve, qPCR can produce an absolute measurement such as number of copies of mRNA per cell. Northern blots, microarrays, Invader assays, and RT-PCR combined with capillary electrophoresis may be used to measure expression levels of mRNA in a sample.

[0138] In some embodiments, nucleic acid amplification methods can be used to detect a polynucleotide biomarker. For example, oligonucleotide primers and probes can be used in amplification and detection methods that use 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, for example the polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), thermophilic SDA (tSDA), Taqman-PCR, multiplex Taqman-PCR, Nanostring, targeted sequencing, digital PCR or any suitable method known in the art. In a preferred embodiment, the level of marker genes can be detected and / or quantified by droplet digital qPCR (ddPCR).

[0139] The detection and quantification of marker genes in the methods of the invention may also involve the use of an agent wherein the agent specifically detects expression products of the marker genes, e.g. proteins or peptides of interest. The agent could be an antibody or functional equivalent thereof that binds proteins or peptides under analysis (i.e. anti-peptide antibody). These antibodies may be used to perform an immunoassay such as, but not limited to, enzyme linked immunosorbent assay (ELISA), radio-immunoassay, immunoprecipitation, immunohistochemistry, immunofluorescence, protein dot blot, Western blot, turbidimetry, nephelometry, FACS, flow cytometry and the like, which are known to the skilled person.

[0140] The relative abundances of the marker genes for the uNK cell subset may be expressed as a ratio e.g. uNKl / uNK3, uNKl / uNK2 or uNK2 / uNK3. Preferably, the relative abundance of the marker genes for uNK3 and uNKl cells are expressed as a ratio, preferably ITGAD / CD160. The relative abundances of the marker genes for decidual cells and decidual senescent cells may be expressed as a ratio, e.g. a PLA2G2A / DIO2 ratio. The fold-change in this ratio provides information regarding the respective levels of these marker genes and may be used in the methods described herein. An increase in the level of marker genes for uNKl cells, and optionally also a decrease in the level of marker genes for uNK3 cells, would lead to an increased ratio. A decrease in the level of marker genes for uNKl cells, and optionally also an increase in the level of marker genes for uNK3 cells, would lead to a decreased ratio. An increase in the level of marker genes for decidual cells, and optionally also a decrease in the level of marker genes for decidual senescent cells, would lead to an increased ratio. A decrease in the level of marker genes for decidual cells, and optionally also an increase in the level of marker genes for decidual senescent cells, would lead to a decreased ratio.

[0141] Method of monitoring or evaluating the effect of a treatment

[0142] The invention further provides a method for monitoring or evaluating the effect of a treatment to reduce the risk (or likelihood or probability) of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effect of the treatment. The relationship between the level of marker genes for uNKl cells and uNK3 cells enables the risk of pregnancy loss or implantation failure to be determined, as described in the preceding section, and thereby allows the determination of whether a treatment is effective in reducing risk. The treatment that may be used to reduce the risk is described further below.

[0143] The marker genes and sample that can be used in the method of monitoring or evaluating the effect of treatment may be any as described for use in the preceding sections.

[0144] Monitoring or evaluating the effect of treatment to reduce the risk of pregnancy loss or embryo implantation failure includes determining whether the individual is responding or has responded to the treatment, determining the nature of the response, determining the extent of the response, and determining whether or not the individual continues to respond to the treatment in the same way over time. In some cases the individual is determined to be responsive to the treatment or to have had a positive response. Responsiveness or a positive response to treatment means that the individual is expected to derive benefit, or a sufficient extent of benefit, as a result of the treatment. For example, the individual may have or be expected to successfully conceive, or the individual may have an improved prognosis. Non-responsiveness or a negative response to treatment means that the individual is not expected to derive benefit, or a sufficient extent of benefit, from receiving the treatment.

[0145] An increased or increasing level of a marker gene for uNKl cells e.g. ITGAD in a sample, as compared with a reference sample or level, may indicate a positive response to treatment. Alternatively, a decreased or decreasing level of a marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, may indicate a positive response to treatment. In one instance, an increased or increasing level of a marker gene for uNKl cells e.g. ITGAD in a sample and a decreased or decreasing level of a marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, together may indicate a positive response to treatment. The relative abundances of the marker genes for uNKl and uNK3 cells may be expressed as a ratio, e.g. ITGAD / CD160 ratio. The fold-change in this ratio may indicate whether treatment is effective. For instance, an increase in the level of marker genes for uNKl cells, and optionally also a decrease in the level of marker genes for uNK3 cells, would lead to an increased ratio, indicating a positive response to treatment by attenuation of decidual senescence.

[0146] A decreased or decreasing or unchanged level of a marker gene for uNKl cells e.g. ITGAD in a sample, as compared with a reference sample or level, may indicate a negative response to treatment. Alternatively, an increased or increasing or unchanged level of the marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, may indicate a negative response to treatment. In one instance, decreased or decreasing or unchanged level of the marker gene for uNKl cells e.g. ITGAD in a sample, and an increased or increasing or unchanged level of the marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, together may indicate a negative response to treatment. There may be a decrease in the ratio of marker genes for uNKl cells to uNK3 cells caused by a decrease in the level of marker genes for uNKl cells, and optionally also an increase in the level of marker genes for uNK3 cells, indicating a negative response to treatment. In one instance, the method for monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual further comprises detecting and / or quantifying the level of uNK cells or the level of at least one marker gene for uNK cells in the sample. The detection and / or quantification of the level of uNK cells, for example based on marker genes may be as described in the preceding sections.

[0147] An increased or increasing level of the uNK cells or uNK cell gene markers in a sample, as compared with a reference sample, may indicate a positive response to treatment. A decreased or decreasing level of uNK cells or uNK cell gene markers, as compared with a reference sample, may indicate a negative response to treatment.

[0148] The above method of treatment of the invention may also further comprise detecting and / or quantifying genes that allow identification of the stage, point or day in the menstrual cycle as described in the preceding sections.

[0149] The control sample or reference sample or level may be selected according to any of the criteria described above. In the method of monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, the level of the marker genes (e.g. ITGAD and CD 160) at a first time point before the treatment may be compared with the level of the marker genes (e.g. ITGAD and CD 160) at a later time point during or after the treatment. The level of the marker genes (e.g. ITGAD and CD 160) during treatment may also be compared with the level of marker genes (e.g. ITGAD and CD 160) at a later time point during or after the treatment. In some instances, the level of marker genes may be determined monthly, bi-monthly, every three months, every four months, every five, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, every twelve months, or at any other suitable time interval as determined by a medical practitioner.

[0150] In a related aspect to the above method, the invention also provides a method of preventing or reducing the risk of pregnancy loss or implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNKl cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNK3 cells in a biological sample obtained from the individual, and administering an agent or carrying out a treatment regimen effective to prevent or reduce the risk of pregnancy loss or implantation failure in the individual. The agent is administered or the treatment regimen carried out if the marker gene levels are indicative of a risk of pregnancy loss or implantation failure as described above.

[0151] Method of diagnosing a reproductive disorder

[0152] The invention additionally provides a method of diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and thereby diagnosing the disorder. The marker genes that can be used in the method of diagnosing a reproductive disorder are as described in the preceding sections.

[0153] The reproductive disorder may be any reproductive disorder. The reproductive disorder may be any disorder associated with infertility, pregnancy loss, miscarriage, associated with the risk of obstetric complications or having a negative impact on pregnancy outcome. The reproductive disorder described herein may be any disorder comprising reduced receptivity or failure of the endometrium to be receptive an embryo. Such disorders may include embryo implantation failure, miscarriage, pregnancy loss, recurrent pregnancy loss or placental disorders. In a preferred embodiment, the reproductive disorder is recurrent pregnancy loss.

[0154] Diagnosis includes determining whether or not the individual has a reproductive disorder. Diagnosis may also include determining the particular cause of the reproductive disorder, and determining the different clinical presentations. A positive diagnosis relates to the determination that an individual has the disorder. A negative diagnosis relates to the determination that an individual does not have the disorder.

[0155] A decreased or decreasing level of a marker gene for uNKl cells e.g. ITGAD in a sample, as compared with a reference sample or level, may indicate a positive diagnosis. Alternatively, an increased or increasing level of the marker genes for uNK3 cells e.g. CD 160, as compared with a reference sample or level, may indicate a positive diagnosis. In one instance, a decreased or decreasing level of the marker gene for uNKl cells e.g. ITGAD in a sample and an increased or increasing level of the marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, together may indicate a positive diagnosis. An unchanged (or similar), increased or increasing level of the marker gene for uNKl cells e.g. ITGAD in a sample, as compared with a reference sample or level, may indicate a negative diagnosis. Alternatively, an unchanged (or similar), decreased or decreasing level of the marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, may indicate a negative diagnosis. In one instance, an unchanged (or similar), increased or increasing level of the marker gene for uNKl cells e.g. ITGAD in a sample, and an unchanged (or similar), decreased or decreasing level of the marker gene for uNK3 cells e.g. CD 160, as compared with a reference sample or level, together may indicate a negative diagnosis.

[0156] In one instance, the method of diagnosing a reproductive disorder in an individual further comprises detecting and / or quantifying the level of uNK cells in general, for example based on the level of at least one marker gene for all uNK cell subsets in the sample. The uNK cell marker genes that may be used in the method of diagnosis may be as described in the preceding sections.

[0157] A decreased or decreasing level of the uNK cells in a sample, as compared with a reference sample, indicates a positive diagnosis. An unchanged (or similar), increased or increasing level of uNK cells, as compared with a reference sample, indicates a negative diagnosis.

[0158] The method of diagnosis of the invention may also further comprise detecting and / or quantifying at least one marker gene for decidual cells, and / or at least one marker for decidual senescent cells.

[0159] The method of diagnosis of the invention may also further comprise detecting and / or quantifying genes that allow identification of the day in the menstrual cycle as described in the preceding sections.

[0160] A control sample or reference sample or level may be provided according to the criteria described above, and may represent a level from an individual or multiple individuals known to have a reproductive disorder, or known to not have any reproductive disorder.

[0161] Methods of therapy

[0162] In accordance with the invention, a method of treating a reproductive disorder in an individual is also provided. The method comprises diagnosing the reproductive disorder according to the method described in the preceding sections and administering an agent or carrying out a treatment regimen effective to treat the reproductive disorder in the individual who is positively diagnosed. Also provided is an agent for use in a method of treating a reproductive disorder in an individual, wherein the reproductive disorder is diagnosed according to the methods described in the preceding sections. Also described is the use of an agent for the preparation of a medicament for the treatment of a reproductive disorder. In some embodiments, the individual has an increased level of at least one marker gene for uNK3 cells e.g. CD 160. In some embodiments, the individual has a decreased level of at least one marker gene for uNKl cells e.g. ITGAD. The term “treating” includes a reduction or prevention of the development or progression of the disorder, and the reduction or elimination of an existing disorder or its symptoms. For instance, an individual may be considered treated if the marker levels are altered such that a negative diagnosis may be made. For instance, the relative abundances of the marker genes for uNKl and uNK3 cells may be expressed as a ratio, e.g. an ITGAD / CD160 ratio. The fold-change in this ratio may indicate whether treatment is effective. For instance, an increase in the level of marker genes for uNKl cells, and optionally also a decrease in the level of marker genes for uNK3 cells, would lead to an increased ratio, indicating a positive response to treatment. There may also be a decrease in the ratio of marker genes for uNKl cells to uNK3 cells caused by a decrease in the level of marker genes for uNKl cells, and optionally also an increase in the level of marker genes for uNK3 cells, indicating a negative response to treatment.

[0163] Agents or treatment regimens that may be administered or carried out may be any agent or treatment regimen known to be effective to treat a reproductive disorder. The agent or treatment regimen may be any able to increase the level of uNKl cells specifically and / or uNK cells in general, and / or decrease the level of uNK3 cells in the individual. Suitable agents or treatment regimens may include but are not limited to endometrial scratching, dipeptidyl-peptidase IV (DPP4) inhibitors (typically gliptins, e.g. sitagliptin), immunomodulatory, immunosuppressive or anti-inflammatory drugs such as corticosteroids and senolytic drugs (e.g. dasatinib, quercetin). Examples of corticosteroids include prednisone and prednisolone. Examples of DPP4 inhibitors include for instance, vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin, evogliptin, omarigliptin, teneligliptin, and are described for example in Deacon CF & Lebovitz HE, Diabetes Obes Metab., 2016;18(4):333-47.

[0164] In some embodiments, the agent is an immunotherapeutic drug (a drug which targets and / or modulates the activity of the immune system in a subject). In some embodiments, the immunotherapeutic drug is human chorionic gonadotropin (hCG), granulocyte-colony stimulating factor (G- CSF), intravenous immunoglobulin (IVIG), or a TNF-alpha inhibitor. The subject may be treated with a single immunotherapeutic drug, a combination of immunotherapeutic drugs, or a combination of at least one immunotherapeutic drug and a further therapy.

[0165] The agent or treatment regimen may target different types of decidual dyshomeostasis, as determined based on the methods of diagnosis previously described. For instance, endometrial scratching may be used to treat decidual failure. Senolytic drugs may be used to treat age-related reproductive disorders. Senolytic drugs or senolytics are drugs that are able to target cellular senescence in order to delay, prevent, alleviate or reverse age-related disorders. The above agents and treatment regimens are also described for use in the methods of reducing the risk of or preventing pregnancy loss or embryo implantation failure described above.

[0166] In some embodiments, the agent is a corticosteroid. In some embodiments, the corticosteroid is prednisolone.

[0167] In a preferred aspect, 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-bound protein and in soluble form (59, 60). DPP4 is also a widely used endometrial receptivity marker gene (61). Stromal cell-derived factor-la (SDF-1), also known as C-X-C motif chemokine ligand 12 (CXCL12), is a potent chemotactic factor that mediates mobilization of BMDC and homing to the endometrium in response to tissue injury and rising oestradiol levels (62, 63). However, SDF-1 is proteolytically inactivated by DPP4. The inventors have identified that DPP4 inhibitors (gliptins), which are commonly used oral antidiabetic drugs for the treatment of type 2 diabetes (64), may 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 e.g. DIO2. DPP4 inhibitors or antagonists may be any agent that inhibits or antagonises DPP4 expression or activity by any means. The agent may inhibit or antagonise inactivation of SDF-1 by DPP4. Such an agent may be a small molecule, a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, small interfering RNA (siRNA) or small hairpin RNA (shRNA) or any other suitable inhibitor that achieves the function described above. The agent may be a polynucleotide encoding a molecule inhibiting or antagonising DPP4 or may be a polynucleotide, oligonucleotide, antisense RNA, siRNA or shRNA inhibiting expression of DPP4, typically comprising a complementary sequence to DPP4 mRNA and specifically hybridising thereto. An oligonucleotide “ specifically hybridises" to a target sequence when it hybridises with preferential or high affinity to the target sequence but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other sequences. More preferably, the oligonucleotide hybridises to the target sequence with a Tmthat is at least 5 °C, at least at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tmfor other nucleic acids. Conditions that permit the hybridisation are well-known in the art (for example, 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-lnterscience, New York (1995)). The hybridisation conditions may be stringent conditions as described in the art.

[0168] The agent may be an antibody that specifically binds to DPP4 protein or to another protein to inhibit DPP4 function indirectly. An antibody “specifically binds” to a protein when it binds with preferential or high affinity to that protein but does not substantially bind, does not bind or binds with only low affinity to other proteins. For instance, an antibody “specifically binds” a target molecule when it binds with preferential or high affinity to that target but does not substantially bind, does not bind or binds with only low affinity to other human proteins.

[0169] An antibody binds with preferential or high affinity if it binds with a Kd of 1 x 10'7M or less, more preferably 5 x 10'8M or less, more preferably 1 x 10'8M or less or more preferably 5 x 10'9M or less. An antibody binds with low affinity if it binds with a Kd of 1 x 10'6M or more, more preferably 1 x 10'5M or more, more preferably 1 x 10'4M or more, more preferably 1 x 10'3M or more, even more preferably 1 x 10'2M or more. 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.

[0170] In a preferred embodiment, the agent used in the method of treatment may be a gliptin, for example including sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin, or dutogliptin. Preferably, the gliptin is sitagliptin.

[0171] In some embodiments, the treatment may include a step of detecting an increased level of a marker gene for uNK3 cells e.g. CD 160.

[0172] Specific routes, dosages and methods of administration of the therapeutic agents described herein may be routinely determined by the medical practitioner. The agents used in the methods of treatment described herein may be formulated in pharmaceutical compositions. These compositions may comprise, in addition to the therapeutically active ingredient(s), a pharmaceutically acceptable excipient, carrier, diluent, buffer, stabilise or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

[0173] The dose may be determined according to various parameters, especially 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, a physician will be able to determine the required route of administration and dosage for any particular patient.

[0174] The agent can be administered to the patient by any suitable means. The agent can be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraosseous, intraperitoneal, intraarticular, topical or other appropriate administration routes. For example, where the agent is a DPP4 inhibitor e.g. sitagliptin, it is preferably administered orally.

[0175] A daily dosage for administration of a gliptin such as sitagliptin to a subject such as a human may range from about 50 mg / day to about 2000 mg / day, such as from about 50 mg / day to about 1500 mg / day, from about 50 mg / day to about 100 mg / day, from about 75 mg / day to about 150 mg / day, from about lOOmg / day to about 1500 mg / day, from about 100 mg / day to about 1200 mg / day, from about 100 mg / day to about 175 mg / day, from about 150 mg / day to about 300 mg / day, from about 200 mg / day to about 350 mg / day, from about 250 mg / day to about 400 mg / day, from about 300 mg / day to about 450 mg / day , from about 350 mg / day to about 500 mg / day, from about 400 mg / day to about 550 mg / day, from about 450 mg / day to about 600 mg / day, from about 500 mg / day to about 750 mg / day, from about 600 mg / day to about 800 mg / day, from about 700 mg / day to about 1000 mg / day, or from about 800 mg / day to about 1200 mg / day. Preferably, a typical daily dose of sitagliptin is about 100 mg / day or at least about 100 mg / day.

[0176] Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, doses can be via a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the agent in the patient and the duration of treatment desired. The dosage as described above may be administered once a day, or may be divided into two doses. The agent may be administered for more than one, for example, at least two or at least three consecutive menstrual cycles. For instance, 100 mg sitagliptin capsules may be taken orally once a day for 2 or 3 consecutive menstrual cycles.

[0177] The method of treatment for medical use may comprise administering additional agents known to be effective in treating reproductive disorders to the individual. For instance, progesterone and / or progestogen may be additionally administered.

[0178] Also provided herein are methods of preventing pregnancy loss or embryo implantation failure, comprising detecting and / or quantifying the amount of at least one marker gene for uNKl cell, at least one marker gene for uNK2 cells and / or at least one marker gene for uNK3 cells in a biological sample obtained from an individual, to thereby assess the individual as being at risk of pregnancy loss or embryo implantation failure, and administering an agent or carrying out a treatment regimen effective to prevent pregnancy loss or embryo implantation failure. Preferably, the at least one marker gene for uNKl cells is ITGAD. Preferably, the at least one marker gene for uNK3 cells is CD 160. The agent or treatment regimen may be any agent or treatment regimen described above, preferably administration of a gliptin such as sitagliptin.

[0179] Also provided are methods for assessing readiness for conception or successful embryo implantation. Such methods also involve detecting and / or quantifying the amount of at least one marker gene for uNKl cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNK3 cells in a biological sample obtained from the individual, and thereby assessing the readiness for conception or embryo implantation. Preferably, the at least one marker gene for uNKl cells is ITGAD. Preferably, the at least one marker gene for uNK3 cells is CD 160. The marker genes and methods of detection that can be used in the above method are as described in the preceding sections.

[0180] Method of selecting patients for treatment

[0181] The invention describes a method of selecting patients for treatment to reduce risk (or likelihood or probability) of embryo implantation failure or pregnancy loss in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNKl cell, at least one marker gene for uNK2 cells and / or at least one marker gene for uNK3 cells in a biological sample obtained from the individual, and thereby selecting the patients for treatment to reduce the risk of pregnancy loss or embryo implantation failure based on the level of the marker genes. Preferably, the at least one marker gene for uNKl cells is ITGAD. Preferably, the at least one marker gene for uNK3 cells is CD160. The method may include detecting and / or quantifying the level of uNK cells in the sample or uNK cell gene markers in a sample. The method may further comprise detecting and / or quantifying the amount of at least one marker gene for decidual cells and / or at least one marker gene for decidual senescent cells, as described above. The marker genes that can be used in the method of selecting patients are as those described in the preceding sections. The treatment may be with any treatment regimen or agent as described above.

[0182] An individual in which an increased level of at least one marker gene for uNK3 cells e.g. CD 160 is detected may be selected as a patient for treatment. An individual in which a decreased level of at least one marker gene for uNKl cells e.g. ITGAD may also be selected as a patient for treatment. The selected patient is preferably treated with a DPP4 inhibitor. Preferably, the marker gene for uNK3 cells is CD160 and the marker gene for uNKl cells is ITGAD, and the selected patient is treated with sitagliptin.

[0183] The method of the invention may also further comprise detecting and / or quantifying genes that allow identification of the day in the menstrual cycle as described in the preceding sections. The relationship between the level of marker genes for uNK cell subsets, decidual cells, decidual senescent cells and uNK cells in general may enable a particular defect in the decidual pathway to be determined. It can then be determined whether a patient would likely benefit from selection of a particular type of treatment to reduce risk of embryo implantation failure or pregnancy loss such as miscarriage.

[0184] Method of stratifying patients

[0185] The invention further provides a method of stratifying patients into different groups, for instance for clinical studies. The method comprises detecting and / or quantifying the amount of at least one marker gene for uNKl cell, at least one marker gene for uNK2 cells and / or at least one marker gene for uNK3 cells in a biological sample obtained from an individual, and thereby stratifying the patients. The method may further comprise detecting and / or quantifying the level of uNK cells in general in the sample for example based on the level of uNK cell gene markers in a sample. The method may further comprise detecting and / or quantifying the amount of at least one marker gene for decidual cells and / or at least one marker gene for decidual senescent cells. The marker genes that may be used in the method of stratifying patients may be any as described in the preceding sections.

[0186] The method of the invention may also further comprise detecting and / or quantifying genes that allow identification of the day in the menstrual cycle as described in the preceding sections.

[0187] The relationship between the level of marker genes for decidual cells, decidual senescent cells and uNK cells may enable the particular defect in the decidual pathway to be determined. Patients having different patterns or levels of these markers can then be grouped accordingly for clinical studies.

[0188] Kits

[0189] The invention further provides a kit, which may be suitable for use in any method of the invention. The kit may include means (e.g. reagents) for detecting and / or quantifying the amount of at least one marker gene for uNKl cell, at least one marker gene for uNK2 cells and / or at least one marker gene for uNK3 cells in a biological sample from an individual The kit thus includes reagents for detecting and / or quantitating said marker genes for uNKl, uNK2, and / or uNK3 cells, preferably reagents for detecting CD 160 and IT GAD. The at least one marker gene for uNK3 cells may comprise CD 160. The kit may further comprise reagents for detecting and / or quantitating marker genes for decidual cells and / or decidual senescent cells. The at least one marker gene for decidual cells may be PLA2G2A. The at least one marker gene for decidual senescent cells may be selected from DIO2, CLU and IGFBP1, preferably DIO2. The kit may also comprise means for detecting and / or quantifying the level of uNK cells or uNK cell markers. The marker genes that may be detected using the kit may be any of those described in the preceding sections. The kit may also further comprise means for detecting and / or quantifying genes that allow identification of the day in the menstrual cycle as described in the preceding sections. Preferably, the genes that allow identification of the timing of the day in the menstrual cycle comprise, consist or consist essentially of GPX3 and SLC15A2. In a preferred embodiment, the kit comprises reagents for detecting and / or quantifying CD 160, ITGAD, PLA2G2A, DIO2, GPX3 and SLC15A2.

[0190] The kit may additionally include instructions for use of the kit in accordance with methods of the invention. The kit may also comprise details regarding which individuals the method may be carried out upon. The kit may also be provided with means for obtaining an endometrial biopsy sample. The kit may also comprise a test requisition form with details to be sent to the analysers. The kit may additionally comprise means for the measurement of other laboratory or clinical parameters, and / or a container for holding a biological sample isolated from a subject.

[0191] The kit may additionally comprise one or more other reagents or instruments which enable the method to be carried out. Such reagents or instruments may include one or more of the following: suitable buffer(s) (aqueous solutions), calibration curve standards, developing reagents, enzymes, labels, reacting surfaces, means for detection, control samples, standards, instructions, interpretive information, means to isolate a relevant biomarker from a sample, means to obtain a sample from the individual (such as a vessel or an instrument comprising a needle) or a support comprising wells on which quantitative reactions can be done.

[0192] In one instance, the kit may comprise a cryotube and RNA stabilizing solution.

[0193] In one instance, use of the above test kit for assessing pregnancy loss or embryo implantation failure, or for diagnosing a reproductive disorder is described. The use may comprise steps as described above in relation to the methods of the invention detecting marker genes. In a preferred embodiment, the kit is used to assessing the risk of, or diagnosing, recurrent pregnancy loss.

[0194] 1. Materials and Methods

[0195] Sample collection

[0196] The study was approved by the NHS National Research Ethics — Hammersmith and Queen Charlotte’s & Chelsea Research Ethics Committee (REC reference: 1997 / 5065) and Tommy’s National Reproductive Health Biobank (REC reference: 18 / WA / 0356). All endometrial biopsies used for this study were obtained from patients attending a dedicated research clinic at University Hospitals Coventry and Warwickshire (UHCW) National Health Service (NHS) Trust, Coventry, UK. Written informed was collected from patients prior to tissue collection in accordance with the guidelines of The Declaration of Helsinki 2000. Endometrial biopsies were timed between 5 and >12 days after the pre-ovulatory LH surge as determined by subject at home urinary analysis.

[0197] Droplet generation and single cell sequencing (Drop-Seq)

[0198] Single-cell transcriptomes were captured in aqueous droplets containing barcoded beads using a microfluidic system (scRNAseq: Dolomite Bio, Royston, UK) according to the manufacturer’s protocol and based on the Drop-Seq method described by Macosko and colleagues (2). Briefly, cells in suspension were placed into the remote chamber of the scRNAseq system. Barcoded beads (Barcoded Bead SeqB; Chemgenes Corp., USA) in lysis buffer at a concentration of 280 beads / pl were loaded into the sample loop. Cell and bead solutions were run at a flow rate of 30 pl / min into a fluorophilic glass microfluidic chip with 100 pm etch depth (Single Cell RNA-seq Chip, Dolomite Bio) with droplet generation oil (Bio-Rad Laboratories, UK) at a flow rate of 200 pl / min for 15-18 minutes. Droplets were collected into a 50 ml Falcon tube, quality checked using a C-Chip Fuchs- Rosenthal Haemocytometer (Labtech, Heathfield, UK), and bead doublets counted.

[0199] Droplet breakage, bead isolation and reverse transcription were performed exactly as described by Macosko and Goldman (Drop-Seq Laboratory Protocol version 3.1, www.mccarrolllab.com / dropseq). All beads from a single run were processed in one batch through Exonuclease I digestion (New England Biolabs UK, Hitchin, UK). PCR was performed on 8000 beads per reaction, with two reactions per sample, to give -800 singlecell transcriptomes attached to microparticles (STAMPS) per timepoint. PCR conditions were as per Macosko and Goldman. Clean-up with Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK) was performed according to standard Illumina RNAseq protocols with a 0.6X beads to sample ratio. cDNA was eluted in 12 pl and quality and size assessed using an Agilent Bioanalyzer High Sensitivity DNA chip. For tagmentation 600 pg cDNA, as determined by Qubit High Sensitivity DNA assay, was processed according to Macosko and Goldman using Illumina Nextera XT DNA Sample Kit and Indexing Kit. Tagmented libraries were cleaned up using AMPure XP beads as before, with a 0.6* beads ratio followed by a repeat clean-up using l x beads. Eluted libraries were analysed using Agilent Bioanalyzer High Sensitivity DNA chip (Agilent, Stockport, UK) to assess quality and determine library size and concentration was determined by Qubit High Sensitivity DNA assay (ThermoFisher, Paisley, UK). Library dilution and denaturation was performed as per standard Illumina protocols and sequenced using NextSeq High Output 75 cycle V2 kit (Illumina, Cambridge, UK).

[0200] Drop-Seq data alignment and quantification Initial Drop-Seq data processing was performed using Drop -Seq tools- 1.0.1 following the protocol described by Nemesh (seqAlignmentCookbook_vl.2Jan2016.pdf, http: / / mccarrolllab.com / dropseq). Briefly, reads with low-quality bases in either cell or molecular barcode were filtered and trimmed for contaminating primer or poly-A sequence. Sequencing errors in barcodes were inferred and corrected, as implemented by Drop-Seq tools-l.O.l. Reads were aligned to the hgl9 (Human) reference genome concatenated with ERCC annotations using STAR-2.5.3a (3), with the Gencode21 (Human) as reference transcriptome. Uniquely mapped reads, with < 1 insertion or deletion, were used in quantification. Finally, the DigitalExpression tool (2) was used to obtain the digital gene expression (DGE) matrix for each sample. Cell numbers were selected computationally from the inflection point in a cumulative distribution of reads plotted against the cell barcodes ordered by descending number of reads. Cell barcodes beyond the inflection point are believed to represent ’ambient RNA’ (e.g. contaminating RNA from damaged cells), not cellular transcriptomes, and therefore excluded from further analysis. This resulted in -800 cells per time-point, matching the number anticipated from processed bead counts. ddPCR

[0201] For digital droplet polymerase chain reaction (ddPCR), a QX200 AutoDG Droplet Digital PCR System (Bio-Rad Laboratories) was employed. Briefly, 10 ng of cDNA was added to a 19 pL reaction mixture containing 1 * supermix for probes (no dUTP), 900 nM target primers / probes, and nuclease-free water. Droplet generation was accomplished using the automated droplet generator (Bio-Rad). The droplet emulsion was subjected to thermal cycling under the following conditions: 95C for 10 minutes, followed by 40 cycles of 94C for 30 seconds and 60C for 1 minute, followed by subsequent enzyme deactivation at 98 for 10 minutes prior to holding at 4C indefinitely. Post-cycling, PCR amplification within the droplets was assessed using the QX200 Droplet Reader (Bio-Rad). Gene expression thresholds were manually set using QXManager software, (version 2.1).

[0202] 2. Discovery of a new biomarker of cytokine uNKl / 2 and cytotoxic uNK3 cells.

[0203] The inventors have previously shown recurrent miscarriage is associated with CD56+ uNK cell deficiency (Brighton et al., 2017, Lucas et al., 2016, Lucas et al., 2020), however little was known how uNK cells modulated this balance. Here, the inventors show that uNK3 (CD160+) cells are involved in the clearance of progesterone resistant stromal cells (Figure 3), whereas uNKl / 2 cells expand as the cycle progresses and confer immune tolerance to the implanting embryo and invading placental cells as well as promote angiogenesis and vascular remodelling. Using high-throughput single-cell droplet barcoding of the transcriptomic changes during the second half of the menstrual cycle (following ovulation) in 12 timed endometrial biopsies from individual patients, we identified four uNK cell subpopulations. (Figure 4a). Proliferating uNK (uNK-P) cells were identified alongside 3 distinct clusters (uNKl / 2 / 3 cells), in keeping with other studies (Strunz et al., 2021, Vento- Tormo et al., 2018).

[0204] Bioinformatic analysis of the scRNA-seq dataset was performed to identify informative biomarker genes. As shown in Figure 4b, CD 160 is a uNK cell specific biomarker gene of cytotoxic uNK3 cells, whilst ITGAD, a gene encoding the a-subunit of P-2 integrin family of membrane glycoprotein (Blythe et al., 2021, Fagerholm et al., 2019), was selectively expressed in immunomodulatory, cytokine producing uNKl / 2 cells. Profiling the relative abundance of uNK subsets across the secretory phase revealed a distinct temporal pattern. In early-secretory endometrium, CD 160+ uNK3 cells were the dominant subpopulation, making up 71.5% of all uNK cells. However, upon the transition to the mid- and late- secretory phase a switch in subpopulation dominance was observed with the expansion of ITGAD+ uNKl / 2 cells in parallel with a relative decline in CD160+ uNK3 cells (Figure 5)

[0205] We created a reference range of expression of both genes in peri-implantation endometrium by digital droplet polymerase chain reaction (ddPCR) analysis of 853 biopsy samples obtained 6 to 11 days after the preovulatory luteinising hormone (LH+6 to LH+11) surge as determined by over-the-counter home ovulation test kits (Figure 6a). Percentiles are used to compare the relative expression of biomarkers in endometrial samples obtained on different days in the menstrual cycle.

[0206] 3. Diagnostic and prognostic potential of normalised ITGAD / CD160 expression ratios in luteal-phase endometrial biopsies in recurrent miscarriage

[0207] Therefore, an essential criterium for any clinical test that purports to identify maternal factors that are causal to miscarriage is that the frequency of a positive test result must increase with each additional pregnancy loss, independently of maternal age. We analysed the ITGAD / CD160 ratio (expressed as percentiles of ratio) in 853 LH-timed endometrial biopsies of women with a history of 0 to 18 prior miscarriages. The lower the ratio of these marker genes, the higher the relative excess of cytotoxic uNK3 cells over cytokine producing uNKl / 2 cells in a sample, and vice versa. As shown in Figure 7A, the frequency of samples with a ratio below the 25th percentile (lower quartile) decreased, whereas the frequency of samples with a ratio above the 75th percentile (upper quartile) increased in a near stepwise with each additional loss (Figure 7B). Importantly, circulating hormone levels for progesterone, thyroid stimulating hormone (TSH) or oestradiol displayed no significant associations with the ITGAD / CD160 ratio percentile (Figure 7C). We also analysed 229 endometrial biopsies obtained prior to a subsequent pregnancy. No restrictions were placed on the time interval between the day of biopsy and the start of the pregnancy. Odds ratios were calculated indicating the likelihood of a subsequent live birth or miscarriage on the basis on ITGAD / CD160 ratios. When all miscarriages were included (unknown karyotyped losses, chromosomally normal (euploid) and chromosomally abnormal (aneuploid) losses), there was no significant association between ITGAD / CD160 ratios in the pre-pregnancy endometrial biopsy and the outcome of a subsequent pregnancy (live birth or miscarriage). However, when aneuploid pregnancy losses were removed from the analysis, ITGAD / CD160 ratios below the 50th percentile were associated with increased risk of a future miscarriage. Conversely, ITGAD / CD160 ratios above the 50th or 75th percentile were associated significantly with increased likelihood of a live birth in a future pregnancy (Figure 7D).

[0208] Example 4 - study of association of both stromal and uNK cell subset markers with pregnancy loss

[0209] The inventors have conducted a prospective study comprising of endometrial samples of 261 women who subsequently became pregnant. The interval between the pre-pregnancy test and subsequent pregnancy was between 1-53 menstrual cycles (median: 4 cycles). In this prospective cohort 177 women had a live birth and 84 experienced another miscarriage. Our of the 84 miscarriages, 46 products of conception (fetal / placental tissues) were successfully karyotyped (genetically analysed). Out of these 46 miscarriages, 28 were ‘euploid’ (no chromosomal abnormalities) and 18 ‘aneuploid’ (chromosomally abnormal). No genetic information was obtained in 38.

[0210] A poor pre-pregnancy decidual reaction (PLA2G2A / DIO2 < 25th percentile) was associated with increased miscarriage risk and decreased likelihood of a live birth in a future conception cycle (OR: 0.52, 95% CI: 0.29-0.92, p = 0.04). This association was more robust upon omission of confirmed aneuploid pregnancy losses from the outcome data (OR: 0.42, 95% CI: 0.23-0.77, p = 0.02). ITGAD / CD160 ratios > 50th percentile favoured live births in a subsequent pregnancy, although statistical significance was only reached upon exclusion of aneuploid losses (OR: 1.89, 95% CL1.05-3.39, p = 0.04). EMBODIMENTS

[0211] 1. A method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uterine natural killer subset 3 (uNK3) cells, at least one marker gene for uterine natural killer subset 2 (uNK2) and / or at least one marker gene for uterine natural killer subset 1 (uNKl) cells in a biological sample obtained from the individual, and thereby assessing the risk.

[0212] 2. The method of embodiment 1, wherein the at least one marker gene for uNKl cells is Integrin Subunit Alpha D (IT GAD).

[0213] 3. The method of embodiment 1 or 2, wherein the at least one marker gene for uNK3 cells is CD160.

[0214] 4. The method according to and m any one of the preceding embodiments, wherein the at least one marker gene for uNKl cells comprises at least one killer immunoglobulin-like receptor (KIR) and / or CD39.

[0215] 5. The method according to any one of the preceding embodiments, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells and at least one marker gene for uNKl cells.

[0216] 6. The method according to any one of the preceding embodiments, wherein the method comprises detecting and / or quantifying the amount of ITGAD and CD160.

[0217] 7. The method according to any one of the preceding embodiments, wherein a decreased level of the marker gene for uNKl cells, as compared with a reference sample or level, and / or an increased level of the marker gene for uNK3 cells, as compared with a reference sample or level, indicates that the individual is at risk of pregnancy loss or embryo implantation failure. The method according to any one of the preceding embodiments, further comprising detecting and / or quantifying the total number or level of uNK cells in the biological sample. The method according to embodiment 8, wherein a decreasing number or level of uNK cells, as compared with a reference sample or level, indicates that the individual is at risk of pregnancy loss or embryo implantation failure. The method according to embodiment 8 or 9, wherein quantifying the number of uNK cells comprises measuring the level of CD56 expression. The method according to any one of the preceding embodiments, wherein the method further comprises a step of determining one or more risk indicia selected from the group consisting of maternal body mass index, maternal age and number of previous miscarriages or embryo implantation failures. A method of monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effect of the treatment. 13. The method of embodiment 12, wherein the at least one marker gene for uNKl cells is Integrin Subunit Alpha D (ITGAD).

[0218] 14. The method of embodiment 12 or 13, wherein the at least one marker gene for uNK3 cells is CD 160.

[0219] 15. The method of any one of embodiments 12-14, wherein the at least one marker gene for uNKl cells comprises at least one killer immunoglobulin-like receptor (KIR) and / or CD39.

[0220] 16. The method of any one of embodiments 12-15, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells and at least one marker gene for uNKl cells.

[0221] 17. The method any one of embodiments 12-16, wherein the method comprises detecting and / or quantifying the amount of ITGAD and CD 160.

[0222] 18. The method any one of embodiments 12-17, wherein

[0223] (i) an increased level of the marker gene for uNK3 cells, as compared with a reference sample or level, and / or a decreased level of the marker gene for uNKl cells, as compared with a reference sample or level, indicates a negative response to treatment; and

[0224] (ii) a decreased level of the marker gene for uNK3 cells, as compared with a reference sample or level, and / or an increased level of the marker gene for uNKl cells, as compared with a reference sample or level, indicates a positive response to treatment.

[0225] 19. The method of any one of embodiments 12-18, wherein the method comprises comparing the level of the marker genes at a first time point before or during the treatment, with the level of the marker genes at a later time point during or after the treatment.

[0226] 20. The method according to any one of the preceding embodiments, wherein the risk of pregnancy loss is risk of miscarriage, and wherein the risk of miscarriage is risk of euploid miscarriage, or wherein the risk of embryo implantation failure is not due to chromosomal abnormalities in an embryo.

[0227] 21. The method according to any one of the preceding embodiments wherein the risk of pregnancy loss is risk of miscarriage, and wherein the risk of miscarriage is risk of recurrent miscarriage.

[0228] 22. A method of diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and thereby diagnosing the individual.

[0229] 23. The method of embodiment 22, wherein the at least one marker gene for uNKl cells is Integrin Subunit Alpha D (ITGAD).

[0230] 24. The method of embodiment 22 or 23, wherein the at least one marker gene for uNK3 cells is CD 160.

[0231] 25. The method of any one of embodiments 22-24, wherein the at least one marker gene for uNKl cells comprises at least one killer immunoglobulin-like receptor (KIR) and / or CD39. 26. The method any one of embodiments 22-25, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells and at least one marker gene for uNKl cells.

[0232] 27. The method any one of embodiments 22-26, wherein the method comprises detecting and / or quantifying the amount of ITGAD and CD160.

[0233] 28. The method according to any one of embodiments 22-27, wherein a decreased level of the uNKl marker genes, as compared with a reference sample or level, and / or an increased level of the uNK3 marker genes, as compared with a reference sample or level, indicates a positive diagnosis.

[0234] 29. The method of any one of the preceding embodiments, wherein the method comprises calculating the ratio of ITGAD expression to CD160 expression.

[0235] 30. The method of embodiment 29, wherein in a positive diagnosis or positive assessment of risk the ratio of ITGAD / CD160 ratio is decreased relative to a reference sample.

[0236] 31. The method of any one of embodiments 22-30, wherein

[0237] (a) in a positive diagnosis or positive assessment of risk, the ratio of ITGAD / CD160 ratio is below the 50thcentile, or below the 25thcentile relative to a reference sample set; and / or

[0238] (b) in a negative diagnosis or negative assessment of risk the ratio of ITGAD / CD160 is above the 50thcentile, or above the 75thcentile relative to a reference sample set.

[0239] 32. The method of any one of the preceding embodiments, wherein the method further comprises a step of determining one or more risk indicia selected from the group consisting of maternal body mass index, maternal age and number of previous miscarriages or embryo implantation failures.

[0240] 33. A method of treating a reproductive disorder in an individual, the method comprising diagnosing the reproductive disorder according to the method of any one of embodiments 22-32, and administering an agent or carrying out a treatment regimen effective to treat the reproductive disorder in the individual who is positively diagnosed.

[0241] 34. An agent for use in a method of treating a reproductive disorder in an individual, the method comprising diagnosing the reproductive disorder according to the method of any one of embodiments 22-32, and administering an agent or carrying out a treatment regimen using the agent effective to treat the reproductive disorder in the individual who is positively diagnosed or assessed as being at risk.

[0242] 35. The method according to embodiment 33 or the agent for use according to embodiment 34, wherein the agent or treatment regimen increases the level of progesterone-responsive stromal cells, and / or decreases the level of progesterone- resistant stromal cells in the individual.

[0243] 36. The method or agent for use according to embodiments 33-35, wherein the agent specifically increases the amount of uNKl cells, and / or decreases the amount of uNK3 cells.

[0244] 37. The method or agent for use according to any one of embodiments 33-36, wherein the agent is a DPP4 inhibitor.

[0245] 38. The method or agent for use according to embodiment 37, wherein the DPP4 inhibitor is sitagliptin. 39. The method or agent for use according to any one of embodiments 33-38, comprising administering progesterone and / or progestogen.

[0246] 40. The method or agent for use according to any one of embodiments 33-39, wherein the reproductive disorder is recurrent miscarriage.

[0247] 41. A method of selecting patients for treatment to reduce risk of embryo implantation failure or pregnancy loss, wherein the method comprises diagnosing the reproductive disorder according to the method of any one of embodiments 22-32, and selecting the patients for treatment to reduce the risk of pregnancy loss or embryo implantation failure based on the level of the marker genes.

[0248] 42. The method according to any one of the preceding embodiments, wherein the biological sample is taken during the luteal phase of the menstrual cycle, optionally wherein the biological sample is taken during the mid-luteal phase of the menstrual cycle.

[0249] 43. The method according to any one of the preceding embodiments, wherein the individual suffers or has suffered from infertility or embryo implantation failure following in vitro fertilisation treatment.

[0250] 44. The method according to any one of the preceding embodiments, wherein the individual has already suffered from at least one previous miscarriage or embryo implantation failure, or is suffering from recurrent pregnancy loss.

[0251] 45. The method according to any one of embodiments 41-44, wherein the patient is selected for treatment with a DPP4 inhibitor.

[0252] 46. The method according to embodiment 45, wherein the DPP4 inhibitor is sitagliptin. The method of any one of the preceding embodiments, wherein the method further comprises detecting and / or quantifying the amount of at least one marker gene for progesterone-responsive stromal cells and at least one marker gene for progesterone-resistant stromal cells in a biological sample obtained from the individual. The method according to embodiment 47, wherein the at least one marker gene for progesterone-responsive stromal cells is selected from PLA2G2A, SCARA5, FTL, GLRX and IL1RL1. The method according to embodiment 47 or 48, wherein the at least one marker gene for progesterone-resistant stromal cells is selected from DIO2, CLU and IGFBP1. The method according to any one of embodiments 47-49, wherein the individual has a level of the at least one marker gene for progesterone-responsive stromal cells, a level of the at least one marker gene for progesterone-resistant stromal cells, and / or a ratio of the level of the at least one marker gene for progesteroneresponsive stromal cells to the level of the at least one marker gene for progesterone-resistant stromal cells, which does not differ significantly from that of a reference sample, optionally wherein the reference sample is from an individual does not have a reproductive disorder and optionally wherein the method comprises assessing the risk of, or diagnosing, silent miscarriage, wherein a positive diagnosis or assessment of risk of silent miscarriage is made when the individual has a ratio of the level of a marker for uNKl cells to the ratio of uNK3 cells which significantly differs from that of a reference sample. The method according to any one of embodiments 47-49, wherein the individual has a level of the at least one marker gene for progesterone-responsive stromal cells, a level of the at least one marker gene for progesterone-resistant stromal cells, and / or a ratio of the level of the at least one marker gene for progesteroneresponsive stromal cells to the level of the at least one marker gene for progesterone-resistant stromal cells, which differs significantly from that of a reference sample, optionally wherein the reference sample is from an individual does not have a reproductive disorder, and optionally wherein the method comprises assessing the risk of, or diagnosing, recurrent miscarriage, wherein a positive diagnosis or assessment of risk of recurrent miscarriage is made when the individual has a ratio of the level of a marker for uNKl cells to the ratio of uNK3 cells which significantly differs from that of a reference sample. The method according to any one of the preceding embodiments, wherein the method further comprises detecting and / or quantifying genes that allow identification of the day in the menstrual cycle, optionally wherein the genes that allow identification of the timing of the day in the menstrual cycle comprise, consist of, or consist essentially of, GPX3 and SLC15A2. The method according to any one of the preceding embodiments, wherein the marker genes are detected and / or quantified using ELISA, Western blotting, immunohistochemistry, immunoassays, enzymatic assays or sequencing methods, optionally wherein the sequencing methods include qPCR, Tagman-PCR, multiplex Tagman-PCR, Nanostring, targeted sequencing or digital PCR. The method according to embodiment 53, wherein the digital PCR is digital droplet PCR (ddPCR). The method according to embodiments 41-54, wherein the method of selecting patients for treatment is to reduce the risk of miscarriage, and wherein the miscarriage is recurrent miscarriage. The method of any one of the preceding embodiments, comprising detecting and / or quantifying the amount of the marker genes in biological samples taken during at least 2 menstrual cycles. 57. A test kit suitable for use in a method of any one of the preceding embodiments, wherein the test kit comprises means for detecting and / or quantifying at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker for uNKl cells in a biological sample obtained from an individual.

[0253] 58. The test kit for use according to embodiment 57, wherein the kit comprises reagents for detecting and / or quantifying IT GAD.

[0254] 59. The test kit for use according to embodiment 57 or 58, wherein the kit comprises reagents for detecting and / or quantifying CD 160.

[0255] 60. The test kit for use according to any one of embodiments 57-59, wherein the kit comprises reagents for detecting and / or quantifying at least one killer immunoglobulin-like receptor (KIR) and / or CD39.

[0256] 61. The test kit for use according to any one of embodiments 57-60, wherein the kit comprises primers for specifically amplifying the sequences of the marker genes by PCR.

[0257] 62. The test kit of any one of embodiments 57-61, wherein the kit comprises reagents for detecting and / or quantifying at least one marker gene for progesteroneresponsive stromal cells and at least one marker gene for progesterone-resistant stromal cells in a biological sample obtained from the individual.

Claims

CLAIMS1. A method for assessing the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uterine natural killer subset 3 (uNK3) cells, at least one marker gene for uterine natural killer subset 2 (uNK2) and / or at least one marker gene for uterine natural killer subset 1 (uNKl) cells in a biological sample obtained from the individual, and thereby assessing the risk.

2. The method of claim 1, wherein:(a) the at least one marker gene for uNKl cells is Integrin Subunit Alpha D (ITGAD); and / or(b) the at least one marker gene for uNK3 cells is CD 160; and / or(c) the at least one marker gene for uNKl cells comprises at least one killer immunoglobulin-like receptor (KIR) and / or CD39; and / or(d) the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells and at least one marker gene for uNKl cells; and / or(e) the method comprises detecting and / or quantifying the amount of ITGAD and CD 160.

3. The method according to claim 1 or 2, wherein a decreased level of the marker gene for uNKl cells, as compared with a reference sample or level, and / or an increased level of the marker gene for uNK3 cells, as compared with a reference sample or level, indicates that the individual is at risk of pregnancy loss or embryo implantation failure.

4. The method according to any one of the preceding claims, further comprising:(a) detecting and / or quantifying the total number or level of uNK cells in the biological sample, optionally wherein:(i) a decreasing number or level of uNK cells, as compared with a reference sample or level, indicates that the individual is at risk of pregnancy loss or embryo implantation failure; and / or(ii) quantifying the number of uNK cells comprises measuring the level of CD56 expression; and / or(b) a step of determining one or more risk indicia selected from the group consisting of maternal body mass index, maternal age and number of previous miscarriages or embryo implantation failures.

5. A method of monitoring or evaluating the effect of a treatment to reduce the risk of pregnancy loss or embryo implantation failure in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and thereby monitoring or evaluating the effect of the treatment.

6. The method of claim 5, wherein:(a) the at least one marker gene for uNKl cells is Integrin Subunit Alpha D (ITGAD); and / or(b) the at least one marker gene for uNK3 cells is CD 160; and / or(c) the at least one marker gene for uNKl cells comprises at least one killer immunoglobulin-like receptor (KIR) and / or CD39; and / or(d) the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells and at least one marker gene for uNKl cells; and / or(e) the method comprises detecting and / or quantifying the amount of ITGAD and CD 160.

7. The method of claims 5 or 6, wherein:(a) an increased level of the marker gene for uNK3 cells, as compared with a reference sample or level, and / or a decreased level of the marker gene for uNKl cells, as compared with a reference sample or level, indicates a negative response to treatment; and(b) a decreased level of the marker gene for uNK3 cells, as compared with a reference sample or level, and / or an increased level of the marker gene for uNKl cells, as compared with a reference sample or level, indicates a positive response to treatment.

8. The method of any one of claims 5-7, wherein the method comprises comparing the level of the marker genes at a first time point before or during the treatment, with the level of the marker genes at a later time point during or after the treatment.

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

10. A method of diagnosing a reproductive disorder in an individual, wherein the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker gene for uNKl cells in a biological sample obtained from the individual, and thereby diagnosing the individual.

11. The method of claim 10, wherein:(a) the at least one marker gene for uNKl cells is Integrin Subunit Alpha D (ITGAD); and / or(b) the at least one marker gene for uNK3 cells is CD 160; and / or(c) the at least one marker gene for uNKl cells comprises at least one killer immunoglobulin-like receptor (KIR) and / or CD39; and / or(d) the method comprises detecting and / or quantifying the amount of at least one marker gene for uNK3 cells and at least one marker gene for uNKl cells; and / or(e) the method comprises detecting and / or quantifying the amount of ITGAD and CD 160; and / or(f) a decreased level of the uNKl marker genes, as compared with a reference sample or level indicates a positive diagnosis; and / or(g) an increased level of the uNK3 marker genes, as compared with a reference sample or level indicates a positive diagnosis; and / or(h) the method comprises calculating the ratio of ITGAD expression to CD 160 expression, optionally wherein in a positive diagnosis or positive assessment of risk the ratio of ITGAD / CD160 ratio is decreased relative to a reference sample; and / or(i) in a positive diagnosis or positive assessment of risk, the ratio of ITGAD / CD160 ratio is below the 50thcentile, or below the 25thcentile relative to a reference sample set; and / or(j) in a negative diagnosis or negative assessment of risk the ratio of ITGAD / CD160 is above the 50thcentile, or above the 75thcentile relative to a reference sample set.

12. The method of any one of the preceding claims, wherein the method further comprises a step of determining one or more risk indicia selected from the groupconsisting of maternal body mass index, maternal age and number of previous miscarriages or embryo implantation failures.

13. A method of treating a reproductive disorder in an individual, the method comprising diagnosing the reproductive disorder according to the method of any one of claims 10-12, and administering an agent or carrying out a treatment regimen effective to treat the reproductive disorder in the individual who is positively diagnosed.

14. An agent for use in a method of treating a reproductive disorder in an individual, the method comprising diagnosing the reproductive disorder according to the method of any one of claims 10-12, and administering an agent or carrying out a treatment regimen using the agent effective to treat the reproductive disorder in the individual who is positively diagnosed or assessed as being at risk.

15. The method according to claim 13 or the agent for use according to claim 14, wherein the agent or treatment regimen increases the level of progesteroneresponsive stromal cells, and / or decreases the level of progesterone-resistant stromal cells in the individual.

16. The method or agent for use according to claims 13-15, wherein:(a) the agent specifically increases the amount of uNKl cells, and / or decreases the amount of uNK3 cells; and / or(b) the agent is a DPP4 inhibitor, optionally wherein the DPP4 inhibitor is sitagliptin; and / or(c) the method comprises administering progesterone and / or progestogen; and / or(d) the reproductive disorder is recurrent miscarriage.

17. A method of selecting patients for treatment to reduce risk of embryo implantation failure or pregnancy loss, wherein the method comprises diagnosing thereproductive disorder according to the method of any one of claims 10-12, and selecting the patients for treatment to reduce the risk of pregnancy loss or embryo implantation failure based on the level of the marker genes.

18. The method according to any one of the preceding claims, wherein:(a) the biological sample is taken during the luteal phase of the menstrual cycle, optionally wherein the biological sample is taken during the mid-luteal phase of the menstrual cycle; and / or(b) the individual suffers or has suffered from infertility or embryo implantation failure following in vitro fertilisation treatment; and / or(c) the individual has already suffered from at least one previous miscarriage or embryo implantation failure, or is suffering from recurrent pregnancy loss.

19. The method according to claim 17 or 18, wherein:(a) the patient is selected for treatment with a DPP4 inhibitor, optionally wherein the DPP4 inhibitor is sitagliptin; and / or(b) the method of selecting patients for treatment is to reduce the risk of miscarriage, and wherein the miscarriage is recurrent miscarriage.

20. The method of any one of the preceding claims, wherein the method further comprises detecting and / or quantifying the amount of at least one marker gene for progesterone-responsive stromal cells and at least one marker gene for progesterone-resistant stromal cells in a biological sample obtained from the individual, optionally wherein:(a) the at least one marker gene for progesterone-responsive stromal cells is selected from PLA2G2A, SCARA5, FTL, GLRX and IL1RL1; and / or(b) the at least one marker gene for progesterone-resistant stromal cells is selected from DIO2, CLU and IGFBP1.

21. The method according to claim 20, wherein:(a) the individual has a level of the at least one marker gene for progesterone-responsive stromal cells, a level of the at least one marker gene for progesterone-resistant stromal cells, and / or a ratio of the level of the at least one marker gene for progesterone-responsive stromal cells to the level of the at least one marker gene for progesterone-resistant stromal cells, which does not differ significantly from that of a reference sample, optionally wherein the reference sample is from an individual does not have a reproductive disorder and optionally wherein the method comprises assessing the risk of, or diagnosing, silent miscarriage, wherein a positive diagnosis or assessment of risk of silent miscarriage is made when the individual has a ratio of the level of a marker for uNKl cells to the ratio of uNK3 cells which significantly differs from that of a reference sample; or(b) the individual has a level of the at least one marker gene for progesterone-responsive stromal cells, a level of the at least one marker gene for progesterone-resistant stromal cells, and / or a ratio of the level of the at least one marker gene for progesterone-responsive stromal cells to the level of the at least one marker gene for progesterone-resistant stromal cells, which differs significantly from that of a reference sample, optionally wherein the reference sample is from an individual does not have a reproductive disorder, and optionally wherein the method comprises assessing the risk of, or diagnosing, recurrent miscarriage, wherein a positive diagnosis or assessment of risk of recurrent miscarriage is made when the individual has a ratio of the level of a marker for uNKl cells to the ratio of uNK3 cells which significantly differs from that of a reference sample22. The method according to any one of the preceding claims, wherein:(a) the method further comprises detecting and / or quantifying genes that allow identification of the day in the menstrual cycle, optionally wherein the genes thatallow identification of the timing of the day in the menstrual cycle comprise, consist of, or consist essentially of, GPX3 and SLC15A2; and / or(b) the marker genes are detected and / or quantified using ELISA, Western blotting, immunohistochemistry, immunoassays, enzymatic assays or sequencing methods, optionally wherein the sequencing methods include qPCR, Tagman-PCR, multiplex Tagman-PCR, Nanostring, targeted sequencing or digital PCR, optionally wherein the digital PCR is digital droplet PCR (ddPCR); and / or(c) the method comprises detecting and / or quantifying the amount of the marker genes in biological samples taken during at least 2 menstrual cycles.

23. A test kit suitable for use in a method of any one of the preceding claims, wherein the test kit comprises means for detecting and / or quantifying at least one marker gene for uNK3 cells, at least one marker gene for uNK2 cells and / or at least one marker for uNKl cells in a biological sample obtained from an individual.

24. The test kit for use according to claim 23, wherein the kit comprises:(a) reagents for detecting and / or quantifying ITGAD, CD 160, at least one killer immunoglobulin-like receptor (KIR) and / or CD39; and / or(b) primers for specifically amplifying the sequences of the marker genes by PCR; and / or(c) reagents for detecting and / or quantifying at least one marker gene for progesterone-responsive stromal cells and at least one marker gene for progesterone-resistant stromal cells in a biological sample obtained from the individual.

Images