Methods for diagnosis of endometriosis

Abstract

A non-invasive method for the diagnosis or prognosis of endometriosis in a subject. The method includes determining the presence of one or more populations of cells in a biological sample, wherein the presence of at least one population of those one or more populations of cells in the biological sample is indicative of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject. In various embodiments of this aspect, the population of cells include subtypes of neutrophils, macrophages, B cells, and / or T cells.

Description

METHODS FOR DIAGNOSIS OF ENDOMETRIOSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of the filing date of U.S. Patent Application Serial No. 63 / 575,567, entitled "Noninvasive Diagnostic for Endometriosis," filed on April 5, 2024, the disclosure of which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01 HD097597 awarded by National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present invention relates to diagnostic methods for identifying endometriosis.BACKGROUND OF THE INVENTION

[0004] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0005] Endometriosis is a chronic gynecological disease that affects an estimated 190 million women (1 in 10 reproductive-aged women) and people who menstruate worldwide. Women with endometriosis experience dysmenorrhea, chronic pain (including dyspareunia, dysuria, and dyschezia), intense pain with menstruation, increased incidence of infertility, and increased risk of systemic and autoimmune diseases as well as associated comorbidities: gastrointestinal dysfunctions, autoimmune disorders, and increased ovarian cancer risk, which dramatically affects their quality of life and reproductive health (See Giudice LC, Kao LC. Endometriosis. Lancet. 2004; 364(9447): 1789-1799; Zondervan KT, et al. Endometriosis. N Engl J Med. 2020; 382(13): 1244-1256; Shigesi N, et al. The association between endometriosis and autoimmune diseases: a systematic review and meta-analysis. Hum Reprod Update. 2019; 25(4): 486-503; and Thomsen LH, et al. Risk factors of epithelial ovarian carcinomas among women with endometriosis: a systematic review. Acta Obstet Gynecol Scand. 2017; 96(6): 761-778).

[0006] These symptoms are caused by the presence of complex, multicellular, uterine-like inflamed lesions that are found outside the uterus. Only the symptoms of endometriosis are treated (e.g., pain medicine, oral contraceptives), and importantly, endometriosis is only definitively diagnosed by invasive surgery. Further, diagnosis is often delayed 7-9 years due to the need for laparoscopic surgical confirmation or incorrect diagnoses, contributing to uncertainty of the true prevalence of endometriosis (Zondervan KT, et al. Endometriosis. N Engl J Med. 2020; 382(13): 1244-1256). If a patient has a blood filled endometrioma on the ovary or a large deep infiltrating lesion, these lesions may be seen by specialized imaging (e.g., trans- vaginal ultrasound, MRI) and suspected as a diagnosis of endometriosis. Unfortunately, these cases account for -30% of cases, leaving 70% of endometriosis undiagnosable by current options.

[0007] Apart from the current issues with an inability to effectively diagnose endometriosis, treatments are mainly palliative with no definitive cure (Rolla E. Endometriosis: advances and controversies in classification, pathogenesis, diagnosis, and treatment. FlOOORes. 2019; 8(529): F1000 Faculty Rev-529). And currently, potential biomarkers for diagnosis lack specificity, leading to ineffective diagnosis and treatment options (See Nisenblat V, et al. Blood biomarkers for the non-invasive diagnosis of endometriosis. Cochrane Database Syst Rev. 2016; 2016(5): Cd012179; and Anastasiu CV, et al. Biomarkers for the noninvasive diagnosis of endometriosis: state of the art and future perspectives. Int J Mol Sci. 2020; 21(5): 1750).SUMMARY OF THE INVENTION

[0008] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0009] One aspect of the present invention is directed to a non-invasive method for the diagnosis or prognosis of endometriosis in a subject. In general, the method includes determining the presence of one or more populations of cells in a biological sample, wherein the presence of at least one population of those one or more populations of cells in the biological sample is indicative of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject. In various embodiments of this aspect, the population of cells include neutrophils (such as, but not limited to, immature inflammatory neutrophils, hyper-segmented (aged) neutrophils, angiogenic neutrophils, inflammatory activated neutrophils, adhesive neutrophils, activated neutrophils, and regulatory anti-apoptotic neutrophils), macrophages (such as, but not limited to, the anti-inflammatoryphagocytic macrophage population), B cells, and / or T cells (such as, but not limited to helper T cells and regulatory T cells). In this respect, one may be concerned with the presence or amount of a particular subpopulation of cells in the subject (e.g., particular subtypes of neutrophils or subtypes of other cell types, such as may be identified by particular biomarkers) as an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

[0010] For example, in one embodiment of the method of this aspect of the present invention, one may obtain a sample of menstrual fluid (or peritoneal fluid) as the biological sample from the subject. One may then determine the particular populations of cells present in the sample (i.e., the various types of cells that are present in the sample). If, for example, one or more particular subtypes of neutrophils are present in the sample of menstrual fluid (or peritoneal fluid), this can be taken as an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

[0011] Alternatively, one may measure the amount of certain subtypes of neutrophils (or other cells of interest that may correlate to endometriosis) that are present in the biological sample, and compare that to a control or reference value. For example, the control or reference value can be the amount of a particular neutrophil subtype relative to total neutrophils that would be compared to that seen in a healthy individual. In one embodiment, the amount of neutrophil subtypes used as the control or reference value is compared to the amount of overall total neutrophils present in the biological sample (i.e. subtypes of neutrophils are corrected for the overall total neutrophil count to not skew data findings). A different amount of the particular subtype or subtypes of neutrophils in the subject (or a particular percentage of change in the level of the subtype or subtypes of neutrophils (or other cell subtype) as compared to the control or reference value) would then indicate the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the general description of the invention given above and the detailed description of the embodiments given below, serve to explain the principles of the present invention.

[0013] FIG. 1 is a pair of images showing samples of menstrual fluid from a healthy individual (image on left) and an individual with endometriosis (image on right). The images show that menstrual fluid from women with endometriosis exhibits hyper-segmented (aged) neutrophils andJhigher numbers of foamy macrophages. Black arrows indicate neutrophils. White arrows indicate macrophages. Arrows with hatching indicate vacuoles.

[0014] FIGS. 2, 2 A, and 2B include a series of graphs regarding neutrophils and certain neutrophil subtypes from healthy women and women with endometriosis. A minimum of six different subpopulations of neutrophils identified in menstrual fluid are shown in the graphs of FIGS. 2A, and 2B as compared to total neutrophils (measured in the graph of FIG. 2 as a ratio of CD45+, CD66b+, CD193’ cells to total CD45+cells). For example, aged (CD45+, CD66b+, CD193’, CD16’ , CXCR2+), classically aged (CD45+, CD66b+, CD193’, CD16’, CXCR2 , CXCR4+), angiogenic aged (CD45+, CD66b+, CD193’, CD16’, CXCR2 , CXCR4+, VEGFR1+, CD54+), inflammatory angiogenic aged (CD45+, CD66b+, CD193’, CD16 , CXCR2", CXCR4+, VEGFR1+, CD54; CXCR3+), early aged angiogenic retained (CD45+, CD66b+, CD193’, CD16+, CXCR4+, CXCR2 , CD49d_, CD62L+, CD274+, CD54hl, VEGFR1+), and early aged adhesive neutrophils (CD45+, CD66b+, CD16+, CD193’, CXCR4+, CXCR210, CD49d", CD62L+, CD18+, CD54+, CD31+, VEGFR1 ) are more abundant in women with endometriosis than healthy women. Letters different from each other represent statistical changes. p<0.05 Two-way ANOVA with Tukey post hoc for multiple comparison analysis.

[0015] FIG. 3 includes two graphs regarding total macrophages (as a ratio of cells expressing CD45 and CD 14 to total cells expressing CD45) and a subpopulation of anti-inflammatory phagocytic macrophages (CD45+, CD1610, CD1410, CD1 lb+, CD1 lc+, VEGFRD, CD31+, CD169+, CD18+, MERTK+) from healthy women and women with endometriosis. The anti-inflammatory phagocytic macrophages showed a marked decrease in women with endometriosis as compared to healthy women. Letters different from each other represent statistical changes. p<0.05 Two-way ANOVA with Tukey post hoc for multiple comparison analysis.

[0016] FIG. 4 is a graph regarding a ratio of B cells (cells expressing both CD45 and CD 19) to total leukocytes (cells expressing CD45) from healthy women and women with endometriosis, showing a decrease in B cells in women with endometriosis as compared to healthy women. Letters different from each other represent statistical changes. p<0.05 Two-way ANOVA with Tukey post hoc for multiple comparison analysis.

[0017] FIG. 5 includes three graphs regarding a ratio of populations of certain T cells (helper T cells CD45+CD3+CD4+CD8"; and regulatory T cells CD45+CD3+CD25+) to total leukocytes (cells expressing CD45) from healthy women and women with endometriosis, showing a decrease in B cells in women with endometriosis as compared to healthy women. Letters different from each other represent statistical changes. p<0.05 Two-way ANOVA with Tukey post hoc for multiple comparison analysis.

[0018] FIGS. 6 A and 6B include images showing samples of menstrual fluid from a healthy individual (“Healthy DI” and “Healthy D2”) and an individual with endometriosis (“Endometriosis DI” and “Endometriosis D2”). The images from FIG. 6A show that menstrual fluid from women with endometriosis exhibits hyper- segmented (aged) neutrophils and higher numbers of foamy macrophages. These figures, then, show the same / similar results as FIG. 1; however, these images are patient specific and show specifics from patient to patient and from day 1 (DI) to day 2 (D2) of menstruation. The hyper- segmented activated neutrophils are present in women with endometriosis and much less present in healthy women. The endometriosis women display a phenotype of activated neutrophils, macrophages, and both cell types having vacuoles present in them in far more quantity than healthy women’s cells. FIG. 6B then further includes images of peritoneal fluid obtained from endometriosis patients undergoing surgery, and finds that the same altered neutrophil phenotype that is found in menstrual fluid is also found in the peritoneal cavity. This suggests that there is a possible systemic altered immune environment; however, it is more likely that the cells from the menstrual fluid are flowing retrograde and are found in the peritoneal cavity which contributes to endometriosis disease.

[0019] FIG. 7 is a table showing Panel A, which is a 28-color spectral flow cytometry panel for identification of neutrophil subtypes, eosinophils, lymphocytes, and endometrial stromal stem cells.

[0020] FIG. 8 is a table showing Panel B, which is a 28-color spectral flow cytometry panel for identification of macrophage subtypes, dendritic cells, and uterine natural killer cells.DETAILED DESCRIPTION OF THE INVENTION

[0021] One or more specific embodiments of the present invention will be described below. To provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system- related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0022] The present disclosure may be understood more readily by reference to the following detailed description of the embodiments taken in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this application is not limited to the specific devices, methods, conditions, or parameters described and / or shown herein,and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting. Also, in some embodiments, as used in the specification and including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” or “approximately” one particular value and / or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0023] While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs.

[0024] One aspect of the present invention involves a noninvasive diagnostic method for identifying endometriosis. As described above in the Background, endometriosis is a chronic gynecological disease that affects an estimated 190 million women and people who menstruate worldwide. Women with endometriosis experience chronic pelvic pain, intense pain with menstruation, increased incidence of infertility, and increased risk of systemic and autoimmune diseases which dramatically affects their quality of life. These symptoms are caused by the presence of complex, multicellular, uterine-like inflamed lesions that are found outside the uterus. Only the symptoms of endometriosis are treated (i.e. pain medicine, oral contraceptives), and importantly, endometriosis is only definitively diagnosed by invasive surgery. If a patient has a blood filled endometrioma on the ovary or a large deep infiltrating lesion, these lesions may be seen by specialized imaging (e.g. trans-vaginal ultrasound, MRI) and suspected as a diagnosis of endometriosis. Unfortunately, these cases account for -30% of cases, leaving 70% of endometriosis undiagnosable by current options.

[0025] In developing various aspects of the present invention, the inventors uncovered a population of neutrophils isolated from menstrual fluid that are more abundant and distinct in women with endometriosis compared to healthy women. The differences, in one aspect of the present invention (and as described in greater detail in Example 1, below), can be elucidated via a simple stain (HEMA 3TM differential stain, Fisher Healthcare™, referred to herein as modified Geimsa) to image the immune cells (which may be isolated immune cells) using a microscope. In this stain, distinct differences are observed that can be read by a researcher, pathologist, or Aldriven computer program. An analogous example would be a pap smear to detect cervical changes that may predict a cervical cancer diagnosis and analogous to Cologuard™ where the sample can be collected at home and sent to a laboratory for analysis. Alternatively, to identify the specific subtype of cells, the isolated cells can be stained with a specific antibody cocktail and flow cytometric analysis would identify the exact neutrophil, macrophage, T cell, or B cell subtypes. This would be analogous to blood tests that identify “B Cell Panel”, “CD45 RA / RO”, “Lymphocyte subpopulations”, or “CD 107 A- NK cell degranulation” that are used to identify immune changes in the peripheral blood. The analysis can be done on menstrual fluid.

[0026] And so, one aspect of the present invention is directed to a non-invasive method for the diagnosis or prognosis of endometriosis in a subject. In general, the method includes determining the presence of one or more populations of cells in a biological sample, wherein the presence of at least one population of those one or more populations of cells in the biological sample is indicative of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject. In various embodiments of this aspect, the population of cells include neutrophils (such as, but not limited to, immature inflammatory neutrophils, hyper-segmented (aged) neutrophils, angiogenic neutrophils, inflammatory activated neutrophils, adhesive neutrophils, activated neutrophils, and regulatory anti-apoptotic neutrophils), macrophages (such as, but not limited to, the anti-inflammatory phagocytic macrophage population), B cells, and / or T cells (such as, but not limited to helper T cells and regulatory T cells). In this respect, one may be concerned with the presence or amount of a particular subpopulation of cells in the subject (e.g., particular subtypes of neutrophils or subtypes of other cell types, such as may be identified by particular biomarkers) as an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

[0027] For example, in one embodiment of the method of this aspect of the present invention, one may obtain a sample of menstrual fluid (or peritoneal fluid) as the biological sample from the subject. One may then determine the particular populations of cells present in the sample (i.e., the various types of cells that are present in the sample). If, for example, one or more particular subtypes of neutrophils are present in the sample of menstrual fluid (or peritoneal fluid), this can be taken as an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

[0028] Alternatively, one may measure the amount of certain subtypes of neutrophils (or other cells of interest that may correlate to endometriosis) that are present in the biological sample, and compare that to a control or reference value. For example, the control or reference value can be the amount of a particular neutrophil subtype relative to total neutrophils that would be comparedto that seen in a healthy individual. In one embodiment, the amount of neutrophil subtypes used as the control or reference value is compared to the amount of overall total neutrophils present in the biological sample (i.e. subtypes of neutrophils are corrected for the overall total neutrophil count to not skew data findings). A different amount of the particular subtype or subtypes of neutrophils in the subject (or a particular percentage of change of level of the subtype or subtypes of neutrophils as compared to the control or reference value) would then indicate the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

[0029] In yet another alternative, one may be concerned with the presence or amount of a particular subpopulation of cells in the subject (e.g., particular types of neutrophils / neutrophils exhibiting particular biomarkers) as an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

[0030] In certain embodiments of this first aspect of the present invention, when determining (a) the presence of one or more populations of cells in the biological sample, or (b) the amount of cells in a population of the one or more populations of cells in the biological sample, the cells determined to be present (or the amount determined) are neutrophils, macrophages, B cells, or T cells - or mixtures thereof. In one particular embodiment, the cells determined to be present, or for which amount is determined, are neutrophils, and the particular subpopulation(s) of neutrophils determined to be present, or for which amount is determined, may be one or more of aged neutrophils, classically aged neutrophils, angiogenic aged neutrophils, inflammatory angiogenic aged neutrophils, early aged angiogenic retained neutrophils, early aged adhesive neutrophils, angiogenic neutrophils, immature inflammatory neutrophils, inflammatory activated neutrophils, adhesive neutrophils, activated neutrophils, and regulatory anti-apoptotic neutrophils.

[0031] In another embodiment, the cells determined to be present, or for which amount is determined, are macrophages, and may be chosen from anti-inflammatory phagocytic macrophages and foamy macrophages.

[0032] In another embodiment, cells determined to be present, or for which amount is determined, are T cells, and may be chosen from helper T cells and regulatory T cells.

[0033] In another embodiment, cells determined to be present, or for which amount is determined, are B cells, and may be at reduced amounts in endometriosis subjects compared to healthy subjects.

[0034] As noted above, the method of the first aspect of the present invention involves determining the presence of a population of cells or determining the amount of cells present. In certain aspects of the present invention this determination (of presence or amount) may be facilitated by determining the status of a cell surface molecule. The status of the particular cell surface moleculeis then indicative of certain cells (such as neutrophils, or macrophages, B cells or T cells) - or of certain subpopulations of one or more cell types. For example, if the cells present in the biological sample include immature inflammatory neutrophils, identification may be made via one or more cell surface molecules and, in the case of immature inflammatory neutrophils, the cell surface molecule of interest may be chosen from CD 16; CD49d+, or combinations thereof.

[0035] In another example, if the cells present in the biological sample include aged neutrophils, identification may be made via one or more cell surface molecules and, in the case of aged neutrophils, the cell surface molecule of interest may be chosen from CD16+, CD49dlo / mid, CXCR2+, CXCR4hl, CD45+, CD66b+, CD193; CD16; or combinations thereof. For instance, in certain embodiments, one may look for a combination of only CD45+, CD66b+, CD 193’, and CD16". However, in other embodiments, one may remove certain cell surface molecules from this combination. This allows one to narrow the number of molecules used when utilizing a process such as flow cytometry to identify presence of a population of cells (such as a particular subtype of neutrophil. This same concept (of varying the combinations of the various cell surface molecules to be identified) applies to the other cell types / subtypes and the various cell surface molecules for each of those types / subtypes.

[0036] In another example, if the cells present in the biological sample include classically aged neutrophils, identification may be made via one or more cell surface molecules and, in the case of classically aged neutrophils, the cell surface molecule of interest may be chosen from CD45+, CD66b+, CD 193; CD 16; CXCR2; CXCR4+, and combinations thereof.

[0037] In another example, if the cells present in the biological sample include angiogenic aged neutrophils, identification may be made via one or more cell surface molecules and, in the case of angiogenic aged neutrophils, the cell surface molecule of interest may be chosen from CD45+, CD66b+, CD 193; CD 16; CXCR2 , CXCR4+, VEGFR1+, CD54+, and combinations thereof.

[0038] In another example, if the cells present in the biological sample include inflammatory angiogenic aged neutrophils, identification may be made via one or more cell surface molecules and, in the case of inflammatory angiogenic aged neutrophils, the cell surface molecule of interest may be chosen from CD45+, CD66b+, CD193; CD16; CXCR2 , CXCR4+, VEGFRD, CD54; CXCR3+, and combinations thereof.

[0039] In another example, if the cells present in the biological sample include early aged angiogenic retained neutrophils, identification may be made via one or more cell surface molecules and, in the case of early aged angiogenic retained neutrophils, the cell surface molecule of interest may be chosen from CD45+, CD66b+, CD 193; CD16+, CXCR4+, CXCR2 , CD49d; CD62L+, CD274+, CD54hl, VEGFR1+, and combinations thereof.

[0040] In another example, if the cells present in the biological sample include early aged adhesive neutrophils, identification may be made via one or more cell surface molecules and, in the case of early aged adhesive neutrophils, the cell surface molecule of interest may be chosen from CD45+, CD66b+, CD16+, CD193’, CXCR4+, CXCR2'°, CD49d", CD62L+, CD18+, CD54+, CD31+, VEGFR I", and combinations thereof.

[0041] In another example, if the cells present in the biological sample include angiogenic neutrophils, identification may be made via one or more cell surface molecules and, in the case of angiogenic neutrophils, the cell surface molecule of interest may be chosen from CD16+, CD49dlo / mid, CXCR2+, CXCR4hi, VEGFR1+, and combinations thereof.

[0042] In another example, if the cells present in the biological sample include inflammatory activated neutrophils, identification may be made via one or more cell surface molecules and, in the case of inflammatory activated neutrophils, the cell surface molecule of interest may be chosen from CD16+CD49dhl, and combinations thereof.

[0043] In another example, if the cells present in the biological sample include adhesive neutrophils, identification may be made via one or more cell surface molecules and, in the case of adhesive neutrophils, the cell surface molecule of interest may be chosen from CDllb+, CDl lc+, CD64+, CD62L+, CD54 / ICAM1+, and combinations thereof.

[0044] In another example, if the cells present in the biological sample include activated neutrophils, identification may be made via one or more cell surface molecules and, in the case of activated neutrophils, the cell surface molecule of interest may be chosen from CD16+, CD49dlo / mid, CXCR2+, CXCR4hi, CD62L10, and combinations thereof.

[0045] In another example, if the cells present in the biological sample include regulatory anti- apoptotic neutrophils, identification may be made via one or more cell surface molecules and, in the case of regulatory anti-apoptotic neutrophils, the cell surface molecule of interest may be chosen from CDl lb+, CDllc+, CD64+, CD274+, and combinations thereof.

[0046] In another example, if the cells present in the biological sample include anti-inflammatory phagocytic macrophages, identification may be made via one or more cell surface molecules and, in the case of anti-inflammatory phagocytic macrophages, the cell surface molecule of interest may be chosen from CD45+, CD1610, CD1410, CDl lb+, CDllc+, VEGFR1+, CD31+, CD169+, CD18+, MERTK+, and combinations thereof.

[0047] In another example, if the cells present in the biological sample include B cells, identification may be made via one or more cell surface molecules and, in the case of B cells, the cell surface molecule of interest may be chosen from CD45+, CD19+, and combinations thereof.

[0048] In another example, if the cells present in the biological sample include helper T cells, identification may be made via one or more cell surface molecules and, in the case of helper Tcells, the cell surface molecule of interest may be chosen from CD45+, CD3+, CD4+, CD8", and combinations thereof.

[0049] In another example, if the cells present in the biological sample include regulatory T cells, identification may be made via one or more cell surface molecules and, in the case of regulatory T cells, the cell surface molecule of interest may be chosen from CD45+, CD3+, CD25+, and combinations thereof.

[0050] As described above, in this aspect of the present invention, one may determine the amount of cells of a particular population of cells in the biological sample, and that amount of cells may be indicative of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject. Further, as described above, in the method, one may compare the amount of cells in a population to a healthy control or reference value. The amount of cells in the particular cell population identified as compared to the amount of the same type of cells that would be present in the healthy control or in the reference value can indicate the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject. For example, in one embodiment, the amount of immature inflammatory neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of immature inflammatory neutrophils is at least two times the amount of immature inflammatory neutrophils in the healthy control or the reference value.

[0051] As described above, the biological sample that may be collected and tested may be menstrual fluid or peritoneal fluid. In particular embodiments where the biological sample is menstrual fluid, the fluid may be collected on Day 1 or Day 2 of the menstrual cycle.

[0052] As described above, one may measure the amount of certain cells (such as subtypes of neutrophils or subtypes of other cells of interest that may correlate to endometriosis) that are present in the biological sample, and compare that to a control or reference value. A different amount of the particular subtype or subtypes of cells in the subject - or a particular percentage or level of change in the amount of the subtype or subtypes of cells (or other cell subtype) as compared to the control or reference value) would then indicate the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject. Measuring and / or determining the amount of certain cells (or subtypes of cells) may include determining a cell count, or it may involve determining a relative amount of cells or subtype of cells in a sample compared to control. So, for example, one may be able to determine based on imaging and viewing images (as in FIG. 1 ) and determined acertain amount of difference (such as a certain increase, like a 2 fold or 3 fold increase in cells in the biological sample as compared to the same type / subtype of cells appearing in the control) without determining an exact amount (or numerical amount).

[0053] In one such embodiment (related to FIG. 1), the amount of hypersegmented neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of hypersegmented neutrophils is at least 1.5 times greater than the amount of hypersegmented neutrophils in the healthy control or the reference value. In another embodiment, the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of hypersegmented neutrophils is at least two times greater than the amount of hypersegmented neutrophils in the healthy control or the reference value. In either of these embodiments, the measurement may be taken on day 1 and / or day 2 of menstruation.

[0054] In another embodiment (related to FIG. 1), the amount of foamy macrophages in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of foamy macrophages is present in at least a 2 to 1 ratio over the amount of foamy macrophages in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and / or day 2 of menstruation.

[0055] In another embodiment (related to FIG. 2), the amount of aged neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of aged neutrophils is at least a 1.5 times change compared to the amount of aged neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 of menstruation.

[0056] In another embodiment (related to FIG. 2), the amount of aged neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if there is an absence of a greater than 1.5 times change compared to the amount of aged neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 2 of menstruation.

[0057] In another embodiment (related to FIG. 2), the amount of classically aged neutrophils in the population of cells in the biological sample may be determined. And an indication of thepresence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of classically aged neutrophils is at least a 2 fold change as compared to the amount of classically aged neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and / or day 2 of menstruation.

[0058] In another embodiment (related to FIG. 2), the amount of angiogenic aged neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of angiogenic aged neutrophils is at least a 1.5 fold change as compared to the amount of angiogenic aged neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and / or day 2 of menstruation.

[0059] In another embodiment (related to FIG. 2), the amount of inflammatory angiogenic aged neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of inflammatory angiogenic aged neutrophils is at least a 1.5 fold change as compared to the amount of inflammatory angiogenic aged neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and / or day 2 of menstruation.

[0060] In another embodiment (related to FIG. 2), the amount of early aged angiogenic neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of early aged angiogenic neutrophils is at least a 1.5 fold change compared to the amount of early aged angiogenic neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 of menstruation.

[0061] In another embodiment (related to FIG. 2), the amount of early aged angiogenic neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of early aged angiogenic neutrophils is at least a 2 fold change compared to the amount of early aged angiogenic neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 2 of menstruation.

[0062] In yet another embodiment regarding early aged angiogenic neutrophils, the amount of early aged angiogenic neutrophils in the population of cells in the biological sample may bedetermined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of early aged angiogenic neutrophils is at least a 1.5 fold change compared to the amount of early aged angiogenic neutrophils in the healthy control or the reference value on day 1 of menstruation, and at least a 2 fold change compared to the amount of early aged angiogenic neutrophils in the healthy control or the reference value on day 2 of menstruation.

[0063] In another embodiment (related to FIG. 2), the amount of early aged adhesive neutrophils in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of early aged adhesive neutrophils is at least a 1.3 fold change compared to the amount of early aged adhesive neutrophils in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and day 2 of menstruation.

[0064] In another embodiment (related to FIG. 3), the amount of anti-inflammatory macrophages in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of anti-inflammatory macrophages is at least a 2 fold change compared to the amount of antiinflammatory macrophages in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and day 2 of menstruation.

[0065] In another embodiment (related to FIG. 4), the amount of B cells in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of B cells is at least a 1.5 fold decrease compared to the amount of B cells in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 and day 2 of menstruation.

[0066] In another embodiment (related to FIG. 5), the amount of helper T cells in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of helper T cells is at least a 1.3 fold increase compared to the amount of helper T cells in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 of menstruation. Further, an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may beconsidered if the amount of helper T cells does not then change from day 1 to day 2 of menstruation compared to the amount of helper T cells in the healthy control or the reference value.

[0067] In another embodiment (related to FIG. 5), the amount of regulatory T cells in the population of cells in the biological sample may be determined. And an indication of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or a correlation to a prognosis of endometriosis in the subject may be considered if the amount of regulatory T cells is at least a 1.5 fold decrease compared to the amount of regulatory T cells in the healthy control or the reference value. In this embodiment, the measurement may be taken on day 1 of menstruation.

[0068] When testing the sample for the presence of cell population(s) (or amounts of cells in population(s)), the method may further include staining the biological sample from the subject (such as by using a modified Geimsa stain). In this method, one may also isolate immune cells from the one or more populations of cells in the biological sample. One may then image the isolated immune cells and observe the isolated immune cells to determine: (a) the presence of one or more populations of cells in the biological sample, or (b) the amount of cells in a population of the one or more populations of cells in the biological sample. Such a particular method will be described in greater detail in the Example, below.

[0069] The method may then further include introducing one or more antibodies to the isolated immune cells from Panel A (FIG. 7) or B (FIG. 8). And, in particular embodiments, the one or more antibodies are antibodies to one or more biomarkers associated with immune cells, wherein the one or more biomarkers are cell surface molecules chosen from, for example, CD3, CD4, CD8, CDl lb, CDl lc, CD14, CD16, CD18, CD19, CD25, CD31, CD45, CD49, CD49d, CD54, CD64, CD62L, CD66b, CD 169, CD 193, CD274, CXCR2, CXCR3, CXCR4, MERTK, VEGFRI, and combinations thereof. The one or more biomarkers may be detected by flow cytometry - and in particular embodiments by spectral flow cytometry.

[0070] The various aspects of the present invention will be further described in, and supported by, the following, non-limiting example.EXAMPLE

[0071] Description / Methods

[0072] The study described in this Example followed a protocol approved by the Institutional Review Board (IRB) at University of Cincinnati (IRB No. 2015-7749). Subjects were recruited via email list-serv through Cincinnati Children’s Hospital and fliers placed throughout the University of Cincinnati Medical campus. Women with laparoscopically diagnosed endometriosis and healthy volunteers without endometriosis were recruited for this study. Inclusion criteria werethe following: age between 18 and 45 years and regular menstrual cycles. A further inclusion criterion for the patients with endometriosis was patient-reported laparoscopically confirmed endometriosis. Endometriosis stage was not asked because patients often do not know their stage of disease. Exclusion criteria for both groups were the use of any hormonal therapy in the preceding three months, having an intrauterine device in place, a history of cancer, current use of immune-modulating medications, and pregnancy. All study participants gave written informed consent. A health history questionnaire was administered to all participants.

[0073] Study participants collected menstrual fluid using a menstrual cup (DivaCup®, Diva International Inc., Ontario, Canada). Menstrual fluid was collected for approximately 6-12 hours on days 1 and 2 of the menstrual cycle. Menstrual fluid was transferred to a sterile 50 mL conical tube and stored by the participants in their refrigerator until acquired by the study team. The samples were transported on ice to the laboratory within 24-48 hours of collection. The cells remained viable for up to 72-96 hours post collection. The observations regarding cell viability are consistent with findings from both Warren et al., Analysis of menstrual effluent: diagnostic potential for endometriosis. Mol Med. 2018 Mar 19;24(1): 1. doi: 10.1186 / s 10020-018-0009-6. PMID: 30134794; PMCID: PMC6016873, and van der Molen et al., Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood. Hum Reprod. 2014 Feb;29(2):303-14. doi: 10.1093 / humrep / det398. Epub 2013 Nov 17. PMID: 24249743.

[0074] Upon receipt of menstrual fluid, volumes from day 1 and day 2 were recorded. The samples were kept on ice and the menstrual fluid was first filtered through sterile gauze to remove mucous and large clots. The fluid was spun at 1200 RPM to separate the cellular from the acellular portion of the menstrual fluid. The cellular portion was diluted with Flow Cytometry Staining (FACS) buffer containing phosphate buffered saline (PBS), 0.5% BSA, 0.1% sodium azide, and 2 mM ethylenediaminetetraacetic acid (EDTA) and filtered through a 70 pm cell strainer. The sample was spun again, resuspended in FACS, and filtered through a 40 pm cell strainer. Each subsequent and sequential filtration ensured removal of epithelial and stromal cells. On the final wash, the cells were resuspended in PBS for density gradient centrifugation to remove red blood cells (RBCs).

[0075] Isolation of immune cells

[0076] Density gradient centrifugation using two layers (bottom layer: density 1.119 g / mL (Histopaque®, Sigma- Aldrich Co.); top layer: density 1.077 g / mL (Lymphoprep™, Alere Technologies) was prepared up to 24 hours prior to menstrual fluid processing to isolate the leukocytes. The Percoll gradient was utilized to reduce neutrophil activation. The menstrual fluid was spun over the density gradient twice to improve the isolation purity of leukocytes from theRBCs. Leukocytes were washed twice with ice cold lx PBS and centrifuged at 1440 RPM (4°C) for 10 minutes. The pellet was resuspended in ice cold FACS buffer. Live cells were identified using Trypan Blue exclusion and counted using a hemacytometer to determine the total number of leukocytes per sample.

[0077] Spectral flow cytometry

[0078] The main difference between spectral flow cytometry and conventional flow cytometry is that spectral flow cytometers capture the entire emission spectrum of the fluorophore creating a detailed signature as opposed to only measuring the peak emission of each fluorophore in a target detector as is the case with conventional flow cytometers. This allows for distinction between fluorophores with very similar but unique overall spectral signatures. Spectral flow cytometry provides flexibility in fluorophore selection and allows for the creation of dynamic, multicolor panels. Spectral flow cytometry was utilized for the present inventors’ experiments.

[0079] Staining Protocols

[0080] Giemsa: Cells were placed onto a positively charged microscope slide at 150,000 / circle using a cytospin (500 RPM for 5 minutes). Cells were allowed to dry and stained using the HEMA 3 manual Giemsa stain following manufacturer’s protocol (Fisher Healthcare™).

[0081] Flow cytometry: The staining protocol and spectral flow cytometry unique panel designs for the study of this Example were developed using the guidelines published in the Optimized Multicolor Immunofluorescence Panel (OMIP) 069: Forty -Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood. The present inventors’ panel design included specific activation markers for white blood cell subtypes, specifically macrophage, neutrophil, and T cells. Cells in FACS buffer were placed in a 96- well U-Bottom plate for staining. One million cells were used for the single stained (SS) reference controls, viability dye, and the unstained samples. Two million cells were used for the multicolor (MC) samples. 50 pL of Fc block master mix containing FACS buffer, 0.2 pL Fc block (Invitrogen™, Cat. No. 14-9161-71), 2.5 pL normal rat serum (Jackson Immunoresearch, Cat. No. 012-000-120), and 2.5 pL normal mouse serum (Jackson Immunoresearch, Cat. No. 015-000-120) was applied to each well containing the SS reference controls and the unstained samples and incubated for 20 minutes on ice. Following Fc blocking, 5 pL of CellBlox™ Blocking Buffer (Invitrogen™, Cat. No. B001T06F01) was applied, and the cells were mixed. This buffer was added to block nonspecific binding of Invitrogen™ NovaFluor™ labels with cells. Cells were stained with each SS reference control antibody from both Panel A and Panel B for 35 minutes on ice in the dark (see FIG. 7 for Panel A and FIG. 8 for Panel B for spectral flow cytometry panels). Following incubation, cells were washed with 100 pL of FACS buffer. Cells were centrifuged (4°C) at 300 x g for 5 minutes. The supernatant was decanted. Cells were resuspended in 200 pLFACS buffer and mixed. Cells were centrifuged (4°C) at 300 x g for 5 minutes. The supernatant was decanted. 100 pL of 1% paraformaldehyde (PFA) in lx PBS was added to all the SS reference controls and unstained samples and incubated for 5 minutes at room temperature. Following incubation, the cells were washed in 100 L of FACS buffer and the cells were centrifuged (4°C) at 300 x g for 5 minutes. The supernatant was decanted, and the cells were washed again in 200 pL of FACS buffer and centrifuged. The supernatant was decanted, and the pellets were resuspended in 150 pL of FACS buffer.

[0082] The MC samples were stained in separate microcentrifuge tubes and added to the 96-well U-Bottom plate following completion of the staining protocol. The MC samples were resuspended in 80 pL of the Fc block master mix and incubated for 20 minutes on ice. Following blocking, the antibodies against chemokine receptors in the panels (CXCR1, CXCR2, CXCR3, CCR3, CX3CR1, CXCR4, and CCR6) were added one at a time in a sequential manner. They were each incubated individually for 10 minutes at room temperature in the dark. Following this incubation, 80 pL of the antibody mix composed of 8 pL of CellBlox™ Blocking Buffer, 10 pL of Super Bright Complete Staining Buffer (Invitrogen™, Cat. No. SB-4401-75), FACS buffer, and either 54.6 pL of antibodies from Panel A or 46.9 pL of antibodies from Panel B. Cells were incubated for 35 minutes on ice in the dark. Following incubation, the cells were washed with 100 pL of FACS buffer and the cells were centrifuged (4°C) at 1200 RPM for 5 minutes. The supernatant was aspirated, and the cells were resuspended in 200 pL FACS buffer. The cells were centrifuged (4°C) at 1200 RPM for 5 minutes and the supernatant was aspirated. 100 pL of 1% PFA in lx PBS was added to each MC sample and incubated for 5 minutes at room temperature. Following incubation, 100 pL of FACS buffer was added. The cells were centrifuged (4°C) at 1200 RPM for 5 minutes and the supernatant was aspirated. The cells were resuspended in 200 pL FACS buffer and centrifuged (4°C) at 1200 RPM for 5 minutes. The supernatant was aspirated, and the samples were resuspended in 150 pL FACS buffer. The MC samples were added back into the 96-well U- Bottom plate and stored at 4°C protected from light until ready to acquire on the spectral flow cytometer within 24 hours.

[0083] Panel design

[0084] Two 28-color spectral flow cytometry panels (Panels A and B, shown in FIGS. 7 and 8, respectively) were developed. Panel A (FIG. 7) contains cell surface markers for identification of neutrophil subtypes, eosinophils, lymphocytes, and endometrial stromal stem cells. Panel B (FIG. 8) contains cell surface markers for macrophage subtypes, dendritic cells, and uterine natural killer cells. Panel optimization was performed using guidelines published by Park et al., Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood. Cytometry A. 10 2020;97(10):1044-1051. doi: 10.I002 / cyto.a.24213,and Ferrer-Font et al, Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry. Curr Protoc. Sep 2021 ; l(9):e222. doi: 10. 1002 / cpz 1.222 (both of which are incorporated by reference herein). Serial antibody dilutions were performed to determine the optimal concentration for each antibody (FIGS. 7 and 8). Cell surface antigens were categorized as primary, secondary, or tertiary and fluorophore brightness (using the stain index) was matched with antigen density. Similarity and Complexity indices were determined for both panels. The Similarity index is a measure of dye pair uniqueness on a scale from 0 to 1. Values close to 0 indicate that the full spectrum signatures of the 2 dyes are very different from each other, and values close to 1 indicate that the signatures are very similar to each other. The Complexity index is an overall measure of uniqueness of all dyes in a full spectrum cytometry panel. The lower the value, the easier it will be to work with the dyes in the panel as the overall spread in the panel will be low. Well-designed large panels (e.g., 35 to 40 colors) will have complexity indices around 40 to 50.

[0085] Next, the present inventors evaluated the SS reference controls to ensure accurate unmixing. Cells were used for the majority of the SS reference controls, but the present inventors found using beads for the following markers resulted in fewer unmixing errors (CD90, CD34, CXCR1, CD54, CD62L, CD49d, CXCR2, CDl lc, VEGFR1). After evaluation and optimization of the SS reference controls, the present inventors confirmed the unmixing accuracy of the MC samples. The next step in optimizing the panel was mitigation of autofluorescence. The menstrual fluid samples were noted to have heterogeneous autofluorescence with two different autofluorescence signatures for the neutrophils / macrophages and the lymphocytes. Once the panels were fully optimized, participant samples were acquired and analyzed.

[0086] Spectral flow cytometry

[0087] Samples were acquired using the 5-laser Cytek Aurora (S10 OD025045) at the Cincinnati Children’s Flow Cytometry Research Core. Deviation from the standard Cytek Assay Settings included changing the forward- scatter (FSC), side-scatter (SSC), and SSC-B parameters to 40-50 for best visualization of the immune cell populations found in menstrual fluid. Events to record was set at 50,000 for SS reference controls and 100,000 for MC samples. Spectral unmixing was performed using FlowJo version 10.7.2. Analysis of flow cytometric data was performed using FlowJo version 10.7.2.

[0088] Gating strategy

[0089] While the present inventors’ sample numbers are low, the findings are already significant (healthy n=18, endometriosis n=13). Tests of subphenotypes of neutrophils were performed using the same protocol described above in this Example. FIGS. 2, 2A, and 2B include a series of graphsshowing the results of these tests of neutrophils and their subpopulations from healthy women and women with endometriosis.

[0090] As can be seen in FIGS. 2, 2A, and 2B, total neutrophil ratio (CD45+CD66b+CD193 ) does not change from control to endometriosis nor from day 1 to day 2 of menstruation. The present inventors then examined subphenotypes of neutrophils based on cell surface markers. In particular, the present inventors examined aged neutrophils (CD45+CD66b+CD193" CD16" CXCR2+) and found that in endometriosis, there are significantly more aged neutrophils on day 1 of menstruation compared to healthy women. On day 2, the cells increase in healthy women, but decrease in women with endometriosis, which suggests misregulation of the cell populations. The present inventors then looked at the classically aged neutrophils (CD45+CD66b+CD193" CD16’ CXCR2 CXCR4+) and found increased populations of these cells in the endometriosis cohort compared to control on day 1 and day 2 of menstruation. The next set of cells tested was the angiogenic aged neutrophils (CD45+CD66b+CD193’ CD16’ CXCR2’ CXCR4+VEGFR1+CD54+). As can be seen in FIGS. 2, 2A, and 2B, these cells show increased levels in women with endometriosis compared to healthy women on days 1 and 2 of menstruation. The inflammatory angiogenic aged neutrophils (CD45+CD66b+CD193' CD16" CXCR2" CXCR4+VEGFR1+CD54" CXCR3+) also increase in endometriosis relative to healthy women corresponding to day of collection (i.e. day 1 vs day 2). The early aged angiogenic retained neutrophils (CD45+CD66b+CD193- CD16+CXCR4+CXCR2’ CD49d" CD62L+CD274+CD54hlVEGFR1+) also increase in endometriosis compared to healthy on day 1 and 2 of menstruation. Finally, the early aged adhesive neutrophils (CD45+CD66b+CD 16+CD 193" CXCR4+CXCR210CD49d' CD62L+CD 18+CD54+CD31+VEGFR 1 ) increase on day 2 of menstruation in endometriosis compared to healthy controls.

[0091] In summary, then, aged, classically aged, angiogenic aged, inflammatory angiogenic aged, early aged angiogenic retained, and early aged adhesive neutrophils are more abundant in women with endometriosis than healthy women.

[0092] Macrophage Results

[0093] Tests of macrophages were performed using the same protocol described above in this Example. FIG. 3 includes two graphs regarding total macrophages and a subpopulation of antiinflammatory phagocytic macrophages from healthy women and women with endometriosis. The anti-inflammatory phagocytic macrophages showed a marked decrease in women with endometriosis as compared to healthy women.

[0094] More specifically, and referring to FIG. 3, total macrophages (CD45+CD14+) exhibit the same pattern in control and endometriosis participants. The change in macrophage is evident in the anti-inflammatory phagocytic macrophage population (CD45+CD1610, CD1410, CDl lb+CDl lc+VEGFR1+CD31+CD169+CD18+MERTK+) which shows a significant decrease inwomen with endometriosis compared to healthy women on day 1 of menstruation, but also close to a decrease in the population on day 2 in endometriosis. Together the findings suggest that the macrophages in endometriosis may have less phagocytic and inflammatory probabilities than do healthy cells of the same type.

[0095] B cell Results

[0096] Tests of B cells were performed using the same protocol described above in this Example. FIG. 4 is a graph regarding a ratio of B cells (cells expressing both CD45 and CD 19) to total leukocytes (cells expressing CD45) from healthy women and women with endometriosis, showing a decrease in B cells in women with endometriosis as compared to healthy women. In particular, B cells (CD45+CD19+) are decreased in women with endometriosis on days 1 and 2 of menstruation. A decrease in B cells could suggest a decrease in antibody production.

[0097] T cell Results

[0098] Tests of T cells were performed using the same protocol described above in this Example. FIG. 5 includes three graphs regarding a ratio of populations of certain T cells (helper T cells CD45+CD3+CD4+CD8"; and regulatory T cells CD45+CD3+CD25+) to total leukocytes (cells expressing CD45) from healthy women and women with endometriosis.

[0099] As can be seen in FIG. 5, total T cells on day 1 of menstruation show increased T cells in endometriosis patients as compared to healthy participants. The differences in the T cell subgroups are in Helper T cells (CD14+CD3+CD4+CD8 ) and Regulatory T cells (CD45+CD3+CD25+). The Helper T cells are increased in endometriosis on day 1 of menstruation. In healthy women an increase in helper T cell recruitment is observed, but this shift in recruitment does not occur in women with endometriosis. The regulatory T cells show a decrease in population in women with endometriosis on day I compared to healthy women. The difference is not apparent at day 2 in healthy compared to endometriosis. Changes in T cells may indicate a reduced ability to respond to antigens and controlling an immune response.

[0100] Finally, for the tests and results described above, the tests show similar results regardless of whether the women are or are not on exogenous hormones (e.g., oral contraceptives).

[0101] Results

[0102] Neutrophil maturation is characterized by visual changes in nuclear features and changes in expression of cell surface markers. Early neutrophils lack nuclear segmentation. As neutrophils age, the size of the cells becomes smaller, the nucleus becomes more segmented, and there is less cytoplasmic RNA. FIG. 1 is a cytospin (stained with HEMA 3TM differential stain, Fisher Healthcare™ or modified Geimsa stain) of menstrual fluid from a healthy subject (image on left in FIG. 1) and an endometriosis subject (image on right in FIG. 1). Healthy women have classically described neutrophils with 2 to 3 nuclear lobs joined together. In contrast, women withendometriosis have neutrophils that are more mature and aged compared to healthy subjects. The neutrophils present in the menstrual fluid of women with endometriosis are multilobed, irregularly shaped, have more lobes than healthy neutrophils, and contained within the nucleus and cytoplasm of these mature neutrophils are clear vacuoles (see arrows in FIG. 1, endometriosis image on right). The significance of these nuclear vacuoles is not known; however, each research subject collected with confirmed endometriosis has this distinct neutrophil phenotype. Women with endometriosis also have larger, more foamy macrophages with irregularly shaped nuclei. In healthy women, the macrophage nuclei are round and the cytoplasm will show some foamy appearance. Visually, a simple test like a traditional pap smear could be developed to diagnose endometriosis from menstrual fluid.

[0103] To confirm the present inventors’ visual observations regarding neutrophils, cell surface markers were used to carefully immunophenotype various immune cell types within menstrual fluid. Based on a series of markers, women with endometriosis have increased aged, classically aged, angiogenic aged, inflammatory angiogenic aged, early aged angiogenic retained, and early aged adhesive neutrophils (FIGS. 2, 2A, and 2B).

[0104] Similarly, changes were seen in certain macrophage, B cell, and T cells. In that regard, the anti-inflammatory phagocytic macrophages showed a marked decrease in women with endometriosis as compared to healthy women. B cells are decreased in women with endometriosis on days 1 and 2 of menstruation. Helper T cells are increased in endometriosis on day 1 of menstruation. And the regulatory T cells show a decrease in population in women with endometriosis on day 1 compared to healthy women.

[0105] One of these readouts (i.e., those shown in FIGS. 1-5, and described above for those figures) may be used to diagnose endometriosis non-invasively through menstrual fluid. For example, one option would be FIG. 1 in which the slides would be read by a pathologist similar to how a pap smear is done and read for a woman at her yearly ob / gyn appointment. For further clarity on the phenotype, the present inventors have included a detailed FIGS. 6A and 6B to show multiple women with endometriosis compared to healthy women. In FIGS 6 A and 6B, peritoneal lavage from endometriosis surgeries shows the same neutrophil phenotype as the menstrual fluid from patients with endometriosis. This suggests that the neutrophil phenotype is systemically altered; however, it is more likely that the menstrual fluid flows retrograde leaving the neutrophil with this unique phenotype in the peritoneal cavity.

[0106] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventiveelements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A non-invasive method for the diagnosis or prognosis of endometriosis in a subject, the method comprising: determining the presence of one or more populations of cells in a biological sample wherein the presence of at least one population of the one or more populations of cells in the biological sample is indicative of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject.

2. The method of claim 1, further comprising: determining the amount of cells in a population of the one or more populations of cells in the biological sample; and comparing the amount of cells in a population to a healthy control or reference value; wherein a change in amount of cells in a population, as compared to the healthy control or the reference value, is indicative of the presence of endometriosis in the subject, or risk of development of endometriosis in the subject, or can be correlated to prognosis of endometriosis in the subject3. The method of claim 1 or claim 2, wherein determining (a) the presence of one or more populations of cells in the biological sample, or (b) the amount of cells in a population of the one or more populations of cells in the biological sample further comprises determining the presence or amount of cells chosen from neutrophils, macrophages, B cells, and T cells.

4. The method of claim 3, wherein the cells determined to be present, or for which amount is determined, are neutrophils, and wherein the neutrophils determined to be present, or for which amount is determined, are chosen from aged neutrophils, classically aged neutrophils, angiogenic aged neutrophils, inflammatory angiogenic aged neutrophils, early aged angiogenic retained neutrophils, early aged adhesive neutrophils, angiogenic neutrophils, immature inflammatory neutrophils, inflammatory activated neutrophils, adhesive neutrophils, activated neutrophils, and regulatory anti-apoptotic neutrophils.

5. The method of claim 3, wherein the cells determined to be present, or for which amount is determined, are macrophages, and wherein the macrophages determined to be present, or for which amount is determined, are chosen from anti-inflammatory phagocytic macrophages and foamy macrophages.

6. The method of claim 3, wherein the cells determined to be present, or for which amount is determined, are T cells, and wherein the T cells determined to be present, or for which amount is determined, are chosen from helper T cells and regulatory T cells.

7. The method of claim 4, wherein determining the presence of a population of cells, or determining the amount of cells present, further comprises determining the status of a cell surface molecule.

8. The method of claim 7, wherein the cells are immature inflammatory neutrophils, and the cell surface molecule is chosen from CD16",CD49d+, and combinations thereof.

9. The method of claim 8, wherein the amount of immature inflammatory neutrophils in the population of cells in the biological sample is determined and is at least two times the amount of immature inflammatory neutrophils in the healthy control or the reference value.

10. The method of claim 7, wherein the cells are aged neutrophils, and the cell surface molecule is chosen from CD45+, CD66b+, CD193", CD16 , CXCR2+and combinations thereof.

11. The method of claim 7, wherein the cells are aged neutrophils, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel A.

12. The method of claim 7, wherein the cells are classically aged neutrophils, and the cell surface molecule is chosen from CD45+, CD66b+, CD193", CD16 , CXCR2 , CXCR4+, and combinations thereof.

13. The method of claim 7, wherein the cells are classically aged neutrophils, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel A.

14. The method of claim 7, wherein the cells are angiogenic aged neutrophils, and the cell surface molecule is chosen from CD45+, CD66b+, CD193", CD16 , CXCR2 , CXCR4+, VEGFR1+, CD54+, and combinations thereof.

15. The method of claim 7, wherein the cells are angiogenic aged neutrophils, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel A.

16. The method of claim 7, wherein the cells are inflammatory angiogenic aged neutrophils, and the cell surface molecule is chosen from CD45+, CD66b+, CD 193", CD 16", CXCR2 , CXCR4+, VEGFR1+, CD54", CXCR3+, and combinations thereof.

17. The method of claim 7, wherein the cells are inflammatory angiogenic aged neutrophils, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel A.

18. The method of claim 7, wherein the cells are early aged angiogenic retained neutrophils, and the cell surface molecule is chosen from CD45+, CD66b+, CD 193", CD16+, CXCR4+, CXCR2 , CD49d', CD62L+, CD274+, CD54hi, VEGFR1+, and combinations thereof.

19. The method of claim 7, wherein the cells are early aged angiogenic retained neutrophils, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel A.

20. The method of claim 7, wherein the cells are early aged adhesive neutrophils, and the cell surface molecule is chosen from CD45+, CD66b+, CD16+, CD193 , CXCR4+, CXCR210, CD49d , CD62L+, CD18+, CD54+, CD31+, VEGFRT, and combinations thereof.

21. The method of claim 7, wherein the cells are early aged adhesive neutrophils, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel A.

22. The method of claim 7, wherein the cells are anti-inflammatory phagocytic macrophages, and the cell surface molecule is chosen from CD45+, CD1610, CD1410, CDllb+, CDllc+, VEGFRT, CD31+, CD169+, CD18+, MERTK+, and combinations thereof.

23. The method of claim 7, wherein the cells are anti-inflammatory phagocytic macrophages, and the cell surface molecule in one of a combination of cell surface molecules, wherein the combination is of cell surface molecules from Panel B.

24. The method of claim 3, wherein the cells are B cells, wherein determining the amount of cells present further comprises determining the status of a cell surface molecule, and the cell surface molecule is chosen from CD45+, CD19+, and combinations thereof.

25. The method of claim 3, wherein the cells are helper T cells, wherein determining the amount of cells present further comprises determining the status of a cell surface molecule, and the cell surface molecule is chosen from CD14+, CD3+, CD4+, CD8", and combinations thereof.

26. The method of claim 3, wherein the cells are regulatory T cells, wherein determining the amount of cells present further comprises determining the status of a cell surface molecule, and the cell surface molecule is chosen from CD45+, CD3+, CD25+, and combinations thereof.

27. The method of claim 1 or claim 2, wherein the biological sample is menstrual fluid or peritoneal fluid.

28. The method of claim 27, wherein the biological sample is menstrual fluid collected on Day 1 or Day 2 of the menstrual cycle.

29. The method of claim 1 or claim 2, further comprising staining the biological sample from the subject.

30. The method of claim 29, wherein a modified Geimsa stain is used in staining the biological sample from the subject.31 . The method of claim 1 or claim 2, further comprising isolating immune cells from the one or more populations of cells in the biological sample.

32. The method of claim 31, further comprising imaging the isolated immune cells, and observing the isolated immune cells to determine (a) the presence of one or more populations of cells in the biological sample, or (b) the amount of cells in a population of the one or more populations of cells in the biological sample.

33. The method of claim 31, further comprising introducing one or more antibodies to the isolated immune cells.

34. The method of claim 33, wherein the one or more antibodies are antibodies to one or more biomarkers associated with immune cells, wherein the one or more biomarkers are cell surfacemolecules chosen from CD3, CD4, CD8, CDl lb, CDl lc, CD14, CD16, CD18, CD19, CD25, CD31, CD45, CD49, CD49d, CD54, CD64, CD62L, CD66b, CD169, CD193, CD274, CXCR2, CXCR3, CXCR4, MERTK, VEGFR1, and combinations thereof.

35. The method of claim 33, wherein the one or more antibodies are antibodies to one or more biomarkers associated with immune cells, wherein the one or more biomarkers is a combination of cell surface molecules from Panel B.

36. The method of claim 34, further comprising detecting the one or more biomarkers by flow cytometry.

37. The method of claim 36, wherein detecting the one or more biomarkers by flow cytometry further comprises detecting the one or more biomarkers by spectral flow cytometry.

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