Compositions and methods for the treatment of endometriosis

Abstract

To provide methods for diagnosis and treatment of endometriosis.SOLUTION: A method comprises: enriching or expanding endometrial stromal cells obtained from a subject; subjecting the enriched or expanded endometrial stromal cells to single-cell processing in microfluidic chambers to produce amplified cDNA; subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from a specific group; diagnosing the subject with endometriosis if expression of the one or more genes is reduced relative to expression of the one or more genes in endometrial stromal cells from a subject without endometriosis; and administering a treatment for endometriosis to the subject diagnosed with endometriosis.SELECTED DRAWING: Figure 2-1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 660,641, filed April 20, 2018, which is incorporated by reference in its entirety.

[0002] Technical field of the invention The present invention relates generally to methods for detecting and diagnosing endometriosis. [Background technology]

[0003] Background of the Invention Endometriosis is one of the most common gynecological diseases in the United States. It is a painful and often debilitating disease affecting more than 6.5 million women aged 15 to 44 in the United States (Buck Louis, et al., Fertil Steril, 96:360-365 (2011)). Endometriosis occurs when the uterine lining grows ectopically, most commonly on the ovaries, fallopian tubes, the tissue that holds the uterus in place, and the outer surface of the uterus. Less commonly, endometrial growths are also found in the vagina, cervix, vulva, intestine, bladder, or rectum. The most common symptoms of endometriosis include pain, bleeding or spotting, infertility, and digestive problems such as diarrhea, constipation, abdominal bloating, or nausea.

[0004] The cause of endometriosis is unknown. Possible causes include retrograde menstrual bleeding, genetic factors, immune system problems, hormonal imbalances, and surgical complications. Because the cause of endometriosis is unknown, current treatments for endometriosis only treat the symptoms, not the disease itself. Furthermore, there is no cure for endometriosis other than surgery to remove endometriotic lesions. Removal of endometriotic lesions is only a temporary treatment option, not a permanent cure for the disease.

[0005] Endometriosis is difficult to diagnose because its symptoms overlap with many other diseases affecting the abdomen and bowel. Diagnosis of endometriosis currently relies on a combination of a clinical history and both invasive and noninvasive testing. Pelvic examination, ultrasound, and MRI are all techniques used to visualize potential endometriotic lesions. However, a definitive diagnosis of endometriosis can currently only be obtained through a type of surgery called laparoscopy, which allows physicians to directly view the endometrial tissue from within the pelvic region. During laparoscopy, physicians can also remove any endometriotic lesions that are discovered. Because laparoscopy is an invasive procedure and expensive, screening for endometriosis is impractical. As a result, many women with endometriosis remain undiagnosed or are incorrectly diagnosed and left untreated. A minimally invasive, early detection method for diagnosing endometriosis is needed.

[0006] It is therefore an object of the present invention to provide a more effective method for diagnosing endometriosis.

[0007] It is also an object of the present invention to provide a minimally invasive method for diagnosing endometriosis. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Buck Louis,et al.,Fertil Steril,96:360~365(2011) Summary of the Invention

[0009] Disclosed herein are methods for diagnosing and treating endometriosis in a patient. Current methods for diagnosing endometriosis are either inefficient or highly invasive. An efficient, non-invasive method for diagnosing endometriosis is provided.

[0010] One embodiment includes enriching or expanding endometrial stromal cells obtained from a subject, subjecting the enriched or expanded endometrial stromal cells to single cell processing in a microfluidic chamber to produce amplified cDNA, and detecting amplified cDNA from a subject. The method further includes the steps of: enriching or expanding endometrial stromal cells obtained from a subject; subjecting the enriched or expanded endometrial stromal cells to single cell processing in a microfluidic chamber to produce amplified cDNA; and detecting amplified cDNA from a subject. The method further includes the steps of: enriching or expanding endometrial stromal cells obtained from a subject; subjecting the enriched or expanded endometrial stromal cells to single cell processing in a microfluidic chamber to produce amplified cDNA from a subject; and detecting amplified cDNA from a subject. The method further includes the steps of: enriching or expanding endometrial stromal cells obtained from a subject; , SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof; diagnosing the subject with endometriosis if expression of the one or more genes is reduced compared to expression of the one or more genes in endometrial stromal cells from a subject without endometriosis; and administering treatment for endometriosis to the subject diagnosed with endometriosis.

[0011] Another embodiment includes enriching or expanding endometrial epithelial cells obtained from a subject, subjecting the enriched or expanded endometrial epithelial cells to single cell processing in a microfluidic chamber to produce amplified cDNA, and detecting amplified cDNA from endometrial epithelial cells containing: DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, PRKACG, PRKC A method is provided for diagnosing and treating endometriosis in a subject in need thereof by subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from the group consisting of A, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof, diagnosing the subject with endometriosis if expression of the one or more genes is elevated compared to expression of the one or more genes in endometrial epithelial cells from a subject without endometriosis, and administering a treatment for endometriosis to the subject diagnosed with endometriosis.

[0012] Yet another embodiment provides a method for enriching or expanding endometrial stromal cells and endometrial epithelial cells obtained from a subject, subjecting the enriched or expanded endometrial stromal cells and endometrial epithelial cells to single cell processing in a microfluidic chamber to produce amplified cDNA, and detecting a gene encoding ... Provided are methods for diagnosing and treating endometriosis in a subject in need thereof by subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from the group consisting of TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof, diagnosing the subject with endometriosis if expression of the one or more genes is decreased compared to expression of the one or more genes in endometrial stromal cells from a subject without endometriosis and increased compared to expression of the one or more genes in endometrial epithelial cells from a subject without endometriosis, and administering treatment for endometriosis to the subject diagnosed with endometriosis.

[0013] Endometrial cells can be obtained from menstrual blood or endometrial biopsy.In one embodiment, stromal cells are isolated by sorting cells using endometrial stromal cell markers CD10, CD146, and CD13.In another embodiment, endometrial epithelial cells are isolated by sorting cells using endothelial epithelial cell markers EpCam+, CD45, and CD9.

[0014] Treatments for endometriosis can be selected from a group including anti-inflammatory drugs, hormone therapy, or surgical removal of the affected tissue.

[0015] In one embodiment, the subject has symptoms of endometriosis. In another embodiment, the subject has previously been diagnosed with endometriosis. [The present invention 1001] 1. A method for diagnosing and treating endometriosis in a subject in need thereof, comprising: enriching or expanding endometrial stromal cells obtained from the subject; subjecting the enriched or expanded endometrial stromal cells to single-cell processing in a microfluidic chamber to produce amplified cDNA; subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from the group consisting of DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, PRKACG, PRKCA, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof; diagnosing the subject with endometriosis if expression of the one or more genes is decreased compared to expression of the one or more genes in endometrial stromal cells from a subject without endometriosis; administering a treatment for endometriosis to said subject diagnosed with endometriosis; A method comprising: [The present invention 1002] 1. A method for diagnosing and treating endometriosis in a subject in need thereof, comprising: enriching or expanding endometrial epithelial cells obtained from the subject; subjecting the enriched or expanded endometrial epithelial cells to single-cell processing in a microfluidic chamber to produce amplified cDNA; subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from the group consisting of DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, PRKACG, PRKCA, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof; diagnosing the subject with endometriosis if expression of the one or more genes is elevated compared to expression of the one or more genes in endometrial epithelial cells from a subject without endometriosis; administering a treatment for endometriosis to said subject diagnosed with endometriosis; A method comprising: [The present invention 1003] 1. A method for diagnosing and treating endometriosis in a subject in need thereof, comprising: enriching or expanding endometrial stromal cells and endometrial epithelial cells obtained from the subject; subjecting the enriched or expanded endometrial stromal and endometrial epithelial cells to single-cell processing in a microfluidic chamber to produce amplified cDNA; subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from the group consisting of DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, PRKACG, PRKCA, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof; diagnosing the subject with endometriosis if expression of the one or more genes is decreased compared to expression of the one or more genes in endometrial stromal cells from a subject without endometriosis and is increased compared to expression of the one or more genes in endometrial epithelial cells from a subject without endometriosis; administering a treatment for endometriosis to said subject diagnosed with endometriosis; A method comprising: [The present invention 1004] The method according to any one of claims 1001 to 1003, wherein the endometrial cells are obtained from menstrual blood. [The present invention 1005] 1004. The method of any one of claims 1001 to 1003, wherein said endometrial cells are obtained from an endometrial biopsy. [The present invention 1006] 1004. The method of any of claims 1001 to 1003, wherein said treatment for endometriosis is selected from the group comprising anti-inflammatory drugs, hormone therapy, or surgical removal of diseased tissue. [The present invention 1007] 1004. The method of any of claims 1001 to 1003, wherein diagnosing said subject with endometriosis further comprises staging said endometriosis. [The present invention 1008] 1007. The method of claim 10, wherein said endometriosis is superficial endometriosis (stage I / II) or deep infiltrating endometriosis (stage III / IV). [The present invention 1009] The method according to any one of claims 1001 to 1003, wherein the subject has symptoms of endometriosis. [The present invention 1010] The method of any of claims 1001 to 1003, wherein said subject has previously been diagnosed with endometriosis. [The present invention 1011] 10. The method of claim 10, wherein said endometriosis is superficial endometriosis (stage I / II) or deep infiltrating endometriosis (stage III / IV). [The present invention 1012] 1002. The method of claim 1001, wherein said endometrial stromal cells are isolated by sorting said cells using endometrial stromal cell markers CD10, CD146, and CD13. [The present invention 1013] 1003. The method of claim 1002, wherein said endometrial epithelial cells are isolated by sorting said cells using endothelial epithelial cell markers EpCam+, CD45, and CD9. [The present invention 1014] The method of the present invention 1001, wherein the one or more genes whose expression is reduced compared to the expression of said one or more genes in endometrial stromal cells from subjects without endometriosis include Cx43, MAPK, TGFBR2, ZO2, and ZO1. [The present invention 1015] The method of the present invention 1001, wherein the one or more genes whose expression is reduced compared to the expression of said one or more genes in endometrial stromal cells from subjects without endometriosis include SNAI1, Twist1, Zeb2, Notch1, VEGFR1, and CD45. [Brief explanation of the drawings]

[0016] [Figure 1-1] Figures 1A-1I show heat maps reflecting the gene expression levels of gap junction genes (connexins), as well as other proteins involved in cell-cell interactions (tight junctions and adherens junctions), and various kinases that regulate them. In the monochrome maps, the highest expression is gray, intermediate expression is black, and lowest expression is white. Each dot corresponds to the expression of a specific gene (arranged in rows) in a specific cell (arranged in columns). Figures 1A-1E show gene expression profiles of enriched stromal cells from endometrial biopsy samples from women with no endometriosis (normal; Figure 1A), early endometriosis (stage I / II; Figure 1B), and more extensive endometriotic lesions in the pelvic cavity (stage III / IV; Figure 1C-1E). These samples were obtained at different stages of the menstrual cycle: ES, early secretory phase (Figure 1A-1C), MS, mid-secretory phase (Figure 1D), and P, proliferative phase (Figure 1E). Figures 1F-1I show gene expression profiles of epithelial cells enriched from endometrial biopsy samples from women with no endometriosis (normal; Figure 1F-G), early endometriosis (stage I / II; Figure 1H), and extensive endometriosis (stage III / IV; Figure 1I). These samples were obtained at different phases of the menstrual cycle: ES, early secretory phase (Figure 1F-H), and P, proliferative phase (Figure 1I). [Figure 1-2]Figures 1A-1I show heat maps reflecting the gene expression levels of gap junction genes (connexins), as well as other proteins involved in cell-cell interactions (tight junctions and adherens junctions), and various kinases that regulate them. In the monochrome maps, the highest expression is gray, intermediate expression is black, and lowest expression is white. Each dot corresponds to the expression of a specific gene (arranged in rows) in a specific cell (arranged in columns). Figures 1A-1E show gene expression profiles of enriched stromal cells from endometrial biopsy samples from women with no endometriosis (normal; Figure 1A), early endometriosis (stage I / II; Figure 1B), and more extensive endometriotic lesions in the pelvic cavity (stage III / IV; Figure 1C-1E). These samples were obtained at different stages of the menstrual cycle: ES, early secretory phase (Figure 1A-1C), MS, mid-secretory phase (Figure 1D), and P, proliferative phase (Figure 1E). Figures 1F-1I show gene expression profiles of epithelial cells enriched from endometrial biopsy samples from women with no endometriosis (normal; Figure 1F-G), early endometriosis (stage I / II; Figure 1H), and extensive endometriosis (stage III / IV; Figure 1I). These samples were obtained at different phases of the menstrual cycle: ES, early secretory phase (Figure 1F-H), and P, proliferative phase (Figure 1I). [Figure 2-1] Figures 2A-2BB show boxplots of single-cell microfluidic PCR expression data for most gap junction genes (indicated by GJ on each plot) in enriched stromal cells (Figures 2A-2N) and epithelial cells (Figures 2O-2BB) from uterine brush biopsies. Patients are identified by numbers along the X-axis of each plot and represent normal, early-stage, and late-stage endometriosis subjects, as shown in Table 3 in the Examples. The Y-axis represents Log10 expression. GJA1 (encoding the Cx43 protein) is the most abundantly expressed connexin in both cell types. However, virtually all connexins show a consistent and significant decrease in expression (indicated by asterisks) with disease progression in stromal cells (Figures 2A-2N) but an increase with disease progression in epithelial cells (Figures 2O-2BB). [Figure 2-2]Figures 2A-2BB show boxplots of single-cell microfluidic PCR expression data for most gap junction genes (indicated by GJ on each plot) in enriched stromal cells (Figures 2A-2N) and epithelial cells (Figures 2O-2BB) from uterine brush biopsies. Patients are identified by numbers along the X-axis of each plot and represent normal, early-stage, and late-stage endometriosis subjects, as shown in Table 3 in the Examples. The Y-axis represents Log10 expression. GJA1 (encoding the Cx43 protein) is the most abundantly expressed connexin in both cell types. However, virtually all connexins show a consistent and significant decrease in expression (indicated by asterisks) with disease progression in stromal cells (Figures 2A-2N) but an increase with disease progression in epithelial cells (Figures 2O-2BB). [Figure 2-3] Figures 2A-2BB show boxplots of single-cell microfluidic PCR expression data for most gap junction genes (indicated by GJ on each plot) in enriched stromal cells (Figures 2A-2N) and epithelial cells (Figures 2O-2BB) from uterine brush biopsies. Patients are identified by numbers along the X-axis of each plot and represent normal, early-stage, and late-stage endometriosis subjects, as shown in Table 3 in the Examples. The Y-axis represents Log10 expression. GJA1 (encoding the Cx43 protein) is the most abundantly expressed connexin in both cell types. However, virtually all connexins show a consistent and significant decrease in expression (indicated by asterisks) with disease progression in stromal cells (Figures 2A-2N) but an increase with disease progression in epithelial cells (Figures 2O-2BB). [Figure 2-4]Figures 2A-2BB show boxplots of single-cell microfluidic PCR expression data for most gap junction genes (indicated by GJ on each plot) in enriched stromal cells (Figures 2A-2N) and epithelial cells (Figures 2O-2BB) from uterine brush biopsies. Patients are identified by numbers along the X-axis of each plot and represent normal, early-stage, and late-stage endometriosis subjects, as shown in Table 3 in the Examples. The Y-axis represents Log10 expression. GJA1 (encoding the Cx43 protein) is the most abundantly expressed connexin in both cell types. However, virtually all connexins show a consistent and significant decrease in expression (indicated by asterisks) with disease progression in stromal cells (Figures 2A-2N) but an increase with disease progression in epithelial cells (Figures 2O-2BB). [Figure 3-1] Figures 3A-3N are box plots showing single-cell microfluidic PCR expression data for several other genes involved in cell-cell interactions other than gap junctions and their regulators (TJP1 = ZO1; Cav = caveolin; CDH2 = N-cadherin; Vim = vimentin; CTNNB = catenin β; PRHACA = protein kinase A; PRKCB = protein kinase C) in enriched endometrial stromal cells (Figures 3A-3G) and endometrial epithelial cells (Figures 3H-3N) from uterine brush biopsies. Graph labeling is as shown in Figures 2A-2BB. [Figure 3-2] Figures 3A-3N are box plots showing single-cell microfluidic PCR expression data for several other genes involved in cell-cell interactions other than gap junctions and their regulators (TJP1 = ZO1; Cav = caveolin; CDH2 = N-cadherin; Vim = vimentin; CTNNB = catenin β; PRHACA = protein kinase A; PRKCB = protein kinase C) in enriched endometrial stromal cells (Figures 3A-3G) and endometrial epithelial cells (Figures 3H-3N) from uterine brush biopsies. Graph labeling is as shown in Figures 2A-2BB. [Figure 4-1]Figures 4A-4H show functional assessment of gap junction intercellular coupling in primary endometrial stromal cells and endometrial epithelial cells from normal and endometriotic subjects. Figure 4A is a schematic diagram illustrating how the assay is performed by loading "donor" cells with a gap junction-permeable dye, dropping them onto a monolayer of recipient cells, and tracking the diffusion of the dye into the monolayer. Figures 4B-4E are representative images showing examples of the assay using stromal cells from a normal subject and an advanced endometriosis patient. Figure 4F is a line graph showing the rate of calcein transfer between donor and recipient cells over time (top graph—normal; bottom graph—endometriosis). The x-axis represents time, and the y-axis represents the number of labeled recipient cells per donor cell. The slope is used as a measure of intercellular coupling efficiency (termed "coupling"). [Figure 4-2] Figures 4A-4H show functional assessment of gap junction intercellular coupling in primary endometrial stromal cells and endometrial epithelial cells from normal and endometriotic subjects. Figures 4G-4H are bar graphs showing the coupling levels of epithelial endometrial cells (Figure 4G) and stromal endometrial cells (Figure 4H) to each other (homotypic coupling - black bars) or to a monolayer of LP9 peritoneal mesothelial cells (heterotypic coupling - gray bars). First, as expected from the expression profiles, stromal cells are less well coupled to each other than epithelial cells. However, when exposed to mesothelial cells (through which endometriosis normally invades), a dramatic increase in coupling is seen, but only in stromal cells from endometriosis patients (significance indicated by paired t-tests). [Figure 5]Figures 5A-5F are fluorescence microscopy images showing Cx43 expression in stromal cells alone (Figures 5A-5C) or exposed to mesothelial cells (Figures 5D-5F) from endometriotic biopsy samples from women without endometriosis (167 normal; Figure 5A, Figure 5D), endometriotic biopsy samples from women with superficial endometriosis (164 endometriosis I-II; Figure 5B, Figure 5E), and endometriotic biopsy samples from women with deep invasive endometriosis (169 endometriosis III-IV; Figure 5C, Figure 5F). Arrows indicate redistribution of Cx43 to the cell surface in endometriotic cells exposed to mesothelial cells, potentially explaining the rapid induction of Cx43 coupling seen in Figures 4A-4H. [Figure 6] Figure 1 shows bar graphs depicting the number of endometrial epithelial and stromal cells that invaded through mesothelial cell monolayers in untreated samples (black bars), samples treated with Cx43 siRNA to reduce cell coupling (gray bars), or samples treated with control siRNA against glutaraldehyde dehydrogenase (GAPDH—hatched bars). Both cell types are invasive, and this invasiveness is nearly eliminated when Cx43 expression is suppressed. However, stromal cells show a significant increase in invasiveness with disease progression, which is dependent on Cx43 expression only in endometriosis samples. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention I. Definition Unless otherwise indicated herein or clearly contradicted by context, use of the terms "a," "an," "the," and similar references in the context of describing the presently claimed invention (particularly in the context of the claims) should be construed to cover both the singular and the plural.

[0018] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein.

[0019] The use of the term "about" is intended to describe a value above or below the stated value by a range of about + / -10%; in other embodiments, values ​​may range above or below the stated value by a range of about + / -5%; in other embodiments, values ​​may range above or below the stated value by a range of about + / -2%; and in other embodiments, values ​​may range above or below the stated value by a range of about + / -1%. The foregoing ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples provided herein, or exemplary language (e.g., "etc."), is intended merely to better clarify the invention and does not impose limitations on the scope of the invention unless specifically claimed. Nothing in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0020] As used herein, "uterus" refers to the organ of the female reproductive system, also known as the womb. The primary function of the uterus is to contain and nourish a fetus until it is ready to be born.

[0021] As used herein, "endometrium" refers to the mucous membrane inside the uterus. The endometrium changes throughout the menstrual cycle. Menstruation is the cyclical shedding of the endometrium in response to hormonal fluctuations. The menstrual cycle is divided into two phases: the follicular or proliferative phase, and the luteal or secretory phase. The proliferative phase is characterized by the development of follicles. The secretory phase generally lasts 14 days and begins after ovulation.

[0022] As used herein, "endometrial cells" refers to cells derived from the endometrium. Endometrial cells can be subdivided into stromal cells and epithelial cells.

[0023] As used herein, "stromal cells" refers to connective tissue cells of any organ. Stromal cells support the function of the parenchymal cells of that organ. Endometrial stromal cells are important in the initiation and maintenance of pregnancy.

[0024] As used herein, "epithelial cells" refers to cells that form a cohesive thin film of cells called epithelium. Epithelial cells function as the outer or inner layer of body surfaces and as the functional unit of secretory glands. Epithelial cells may be specialized to function as absorptive, secretory, or barrier.

[0025] As used herein, "endometriosis" refers to a gynecological disease in which tissue from the uterus grows within the abdominal cavity outside the uterus. Two major symptoms of endometriosis include pain and infertility. The primary causes of pain and infertility are endometrial implants and adhesions. As used herein, "endometrial implants" refer to endometrial tissue found in ectopic sites. Implants resemble small, flat patches on the peritoneal surface of the pelvic region. These implants can cause irritation and inflammation in surrounding tissues, leading to the formation of adhesions. As used herein, "adhesions" refer to bands of internal scar tissue that can attach to normally mobile tissues and organs.

[0026] Endometriosis is classified into "stages" based on the severity of the disease, the extent of the disease's spread, the involvement of pelvic structures, the extent of pelvic adhesions, and blockage of the fallopian tubes. The staging of endometriosis does not necessarily reflect the severity of symptoms experienced by the patient.

[0027] The four stages of endometriosis correspond to minimal, mild, moderate, and severe. Most patients fall into the minimal and mild ranges. The mildest endometriosis, called stage I, is characterized by isolated implants and the absence of significant adhesions. Mild endometriosis, stage II, is characterized by superficial implants less than 5 cm in size in clumps without significant adhesions. Stages I and II endometriosis are often combined into the same category, called "superficial endometriosis." Moderate (stage III) endometriosis is characterized by the appearance of endometriomas, a type of cyst that forms when endometrial tissue grows in the ovaries. Severe endometriosis (stage IV) is characterized by multiple implants, cysts, and severe adhesions that result in scarring around the fallopian tubes and ovaries. Women with stage IV endometriosis are most likely to have infertility problems.

[0028] As used herein, "gap junction" refers to an organized collection of protein channels in cell membranes that allow the passage of ions and small molecules between adjacent cells.

[0029] II. Methods for diagnosing and treating endometriosis The present specification provides a method for diagnosing and treating endometriosis. The present specification discloses a highly sensitive single-cell expression analysis method for detecting specific gene expression patterns in a cell population obtained from endometrial cells. An exemplary method includes enriching or expanding endometrial cells obtained from a subject, subjecting the cells to single-cell processing in a microfluidic chamber to produce amplified cDNA, and detecting the following genes: DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, P The method includes subjecting the amplified cDNA to microfluidic PCR to detect RNA gene expression of one or more genes selected from the group consisting of RKACG, PRKCA, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, ZO2, and combinations thereof; diagnosing the subject with endometriosis if expression of the one or more genes is elevated compared to expression of the one or more genes in endometrial epithelial cells from a subject without endometriosis; and administering treatment for endometriosis to the subject diagnosed with endometriosis.

[0030] A. Enrichment and proliferation of endometrial cells 1. Endometrial Sample In one embodiment, a sample of uterine cells is collected from a woman during a routine health check. In some embodiments, the woman is suspected of having endometriosis. In another embodiment, the woman has previously been diagnosed with endometriosis, and the disclosed methods are used to monitor for recurrence of the disease.

[0031] In one embodiment, endometrial cells are obtained from a subject non-invasively, for example, in menstrual fluid collected with a Stericup. In another embodiment, endometrial cells are obtained from a subject with a minimally invasive method, such as an endometrial brush biopsy device or an endometrial aspiration catheter.

[0032] Women with endometriosis may have superficial endometriosis (stage I / II) or deep infiltrating endometriosis (stage III / IV). Biopsy specimens can be obtained at different stages of the menstrual cycle, including, but not limited to, the early secretory phase, mid-secretory phase, or proliferative phase.

[0033] Some embodiments provide methods for subjecting uterine cells to single-cell processing and microfluidic PCR. The uterine cells can be stromal or epithelial cells.

[0034] Endometrial biopsy specimens can be prepared for enrichment, proliferation, and single-cell processing by isolating cells from tissue.Methods for isolating cells from endometrial biopsy specimens are known in the art.Exemplary methods include but are not limited to collagenase digestion, trypsin digestion, and manual scraping of the surface epithelium from the whole biopsy specimen (Krjutskov, K., et al. Human Reproduction, 31:844-853 (2016); Jividen, K., et al., J Vis Exp, 87:e51513).

[0035] The menstrual fluid is prepared for concentration, expansion, and single cell processing by removing red blood cells from the fluid and isolating endometrial cells.

[0036] 2. Cell enrichment In one embodiment, the endometrial cell population from biopsy material is enriched with stromal cells or epithelial cells.Methods for enriching endometrial cell population with stromal cells or epithelial cells are known in the art.Exemplary methods include, but are not limited to, physical separation using filtration devices, flow cytometry, magnetic beads or microbeads coated with specific antibodies, and microfluidics.Enriched samples can be grown in culture before single cell processing.

[0037] i.Filtration In one embodiment, stromal cells are isolated from the endometrial cell population by passing the digested cell suspension through a cell culture filter. The stromal cells pass through the filter, while the epithelial cells remain aggregated within the tissue, which does not pass through the filter. The epithelial tissue can be further digested into a single cell suspension. The concentrated cell suspension can be cultured for expansion or used directly for single cell processing.

[0038] ii.FACS Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry with sorting capabilities capable of isolating single cells. Prior to separation, a cell suspension is created and target cells are labeled with a fluorescent probe. Fluorophore-conjugated monoclonal antibodies (mAbs) are the most widely used fluorescent probes, recognizing specific surface markers on target cells. As the cell suspension passes through the device, each cell is exposed to a laser, allowing a fluorescence detector to identify the cell based on selected properties, specifically which antibody is bound. The instrument applies an electric charge (positive or negative) to droplets containing cells of interest, and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes for subsequent analysis. FACS can also be used to sort single cells.

[0039] In one embodiment, cells from endometrial biopsy specimens are sorted using FACS. The cell suspension from endometrial biopsy specimens can be incubated with fluorescent probes that recognize stromal cell markers, such as CD10, CD146, and CD13. The cell suspension is run through a flow cytometer, and stromal cells are collected in a separate container. In another embodiment, the cell suspension is incubated with fluorescent probes that recognize epithelial markers, such as EpCam+ and CD9. The cell suspension is run through a flow cytometer, and epithelial cells are collected in a separate container. In one embodiment, endometrial cell cultures are enriched for stromal or epithelial cells using FACS.

[0040] iii. Magnetic beads Magnetic bead cell isolation is a technique used to enrich specific cell types from a mixed population of cells. Magnetic beads or nanoparticles conjugated with antibodies against cell surface markers on target cells are mixed with the cell population. The container holding the cells is exposed to a magnetic column, and the cells of interest (conjugated with magnetic beads) are separated from the rest of the cells. In one embodiment, magnetic beads conjugated with antibodies against stromal cell markers such as CD10, CD146, and CD13 are used to separate stromal cells from an endometrial cell suspension.

[0041] iv. Microfluidics In another embodiment, cells from endometrial biopsies are enriched using microfluidics. Cell sorting by microfluidic chips can be divided into four categories: microfluidic separation based on cell affinity chromatography, microfluidic separation based on the physical properties of cells, microfluidic separation based on immunomagnetic beads, and separation methods based on the differences in dielectric properties between various cell types.

[0042] Microfluidics based on cell affinity chromatography is the most commonly used method for microfluidic chip analysis. It relies on highly specific interactions between antigens and antibodies, or ligands and receptors. At the beginning of processing, microchannels within the chip are modified with specific antibodies that can bind to cell surface antigens or aptamers. As the sample flows through the microchannels, the cell surface antigens can bind to specific antibodies or aptamers that immobilize cells on the chip, while the remaining cells are flushed out of the chip by a buffer solution. Finally, a different buffer solution can be used to elute the immobilized cells for downstream analysis. In one embodiment, an endometrial cell population is enriched for stromal cells using microfluidics. In another embodiment, epithelial cells are enriched.

[0043] 3. Cell Growth In other embodiments, cells are grown in culture without enrichment for specific cell subtypes. Endometrial biopsies can be prepared for cell culture growth by isolating cells from tissue. Methods for isolating cells from endometrial biopsies are known in the art. Exemplary methods include, but are not limited to, collagenase digestion, trypsin digestion, and manual scraping of the surface epithelium from the entire biopsy (Krjutskov, K., et al. Human Reproduction, 31:844-853 (2016); Jividen, K., et al., J Vis Exp, 87:e51513). Methods for culturing endometrial cells are known in the art. See, e.g., Osteen, KG, et al., Fertility and Sterility, 52:966-972 (1989); Zhang, L., et al., J Cell Sci, 108:323-331 (1995). In some embodiments, the high sensitivity of single-cell PCR overcomes the need to grow cells in culture prior to analysis.

[0044] B. Single Cell Processing In one embodiment, the enriched or expanded stromal cells or uterine cells are subjected to single-cell processing. Single-cell processing is a method for isolating a single cell from a cell population and performing analysis on a single cell rather than the entire cell population. In one embodiment, a single cell from an endometrial sample is subjected to microfluidic PCR.

[0045] 1. Single Cell Isolation In one embodiment, stromal cells are isolated from endometrial cell culture.Exemplary endometrial stromal cell markers include but are not limited to CD10, CD146 and CD13.In another embodiment, epithelial cells are isolated from endometrial cell culture.Exemplary epithelial markers include but are not limited to EpCam+, CD45 and CD9.

[0046] Methods for isolating single cells from whole cell cultures are known in the art. Exemplary methods for isolating single cells from large populations of cells include manual cell harvesting, flow cytometry, magnetic-activated cell sorting, and microfluidics.

[0047] Methods for isolating rare cells from a population or single cells from a small sample are also known in the art. Dielectrophoresis (DEP) microfluidic systems use a microfluidic chip equipped with a dielectrophoretic cage to navigate individual cells by electrical charge after identifying them with a fluorescent marker. The advantage of these systems is that all cells are preserved, and even single cells within a pool of 100,000 can be efficiently isolated. An exemplary DEP system is the DEP-Array™ system (Silicon Biosciences). In one embodiment, endometrial cells are separated into single-cell samples using a DEP system. Cells can be labeled with stromal or epithelial markers to separate the two cell populations. The DEP system can distribute each single cell into individual wells of a microplate for further processing.

[0048] 2. Microfluidic PCR In one embodiment, the processed single-cell-derived cDNA is used in microfluidic PCR. Microfluidics is a miniaturized device that can process samples containing large amounts of fluid, on the order of nanoliters or picoliters. Microfluidic PCR systems can successfully detect nucleic acid expression from nanoliter-sized samples. In one embodiment, the microfluidic PCR device is a single-cell microfluidic PCR device. Examples of commercially available microfluidic PCR devices include the BioMark™ HD Single Cell System (Fluidigm) or the C1™ System (Fluidigm).

[0049] i. Gap junctions Gap junctions are special intercellular connections between cells. These connections, or channels, provide direct intercellular communication between the cytoplasm of two cells, allowing for the rapid exchange of ions and metabolic products up to approximately 1 kD in size. Gap junctions are formed from clusters of connexin proteins. Exemplary gap junction genes include, but are not limited to, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, and GJC2. In one embodiment, expression of gap junction genes is elevated in endometrial epithelial cells but decreased in endometrial stromal cells from women with endometriosis.

[0050] Intercellular gap junction communication has been investigated as a mode of intercellular communication between endometrial cells and the mesothelium, the invasive target of endometriosis. Using conventional cell assays and single-cell analysis, specific gap junction proteins (channel-forming connexins) that may be involved in endometrial cell invasiveness leading to the development of endometriotic lesions have been identified. In addition to gap junction genes, other regulatory genes and kinases are involved in gap junction communication. Exemplary kinases that regulate gap junctions include, but are not limited to, tyrosine kinase, protein kinase C, cAMP-dependent protein kinase, MAP kinase, cdc2 / cyclin B, and casein kinase I (Warn-Cramer, BJ, and Lau, AF, Biochim Biophys Acta, 1662:81-95 (2004); Lampe, PD, and Lau, AF, Int J Biochem Cell Biol, 36:1171-1186 (2004)).

[0051] In one embodiment, RNA gene expression of DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, PRKACG, PRKCA, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, and ZO2 in cDNA from a single cell sample is measured by microfluidic PCR.

[0052] In another embodiment, the expression level of one of the disclosed genes in a sample is compared to the expression level of the same gene in a sample from a healthy individual without endometriosis.

[0053] C. Diagnosis of endometriosis In the disclosed methods, DBN1, CAV1, CDH, CDK1, CD45, CK19, CSNK, CTNNB1, Cx43, EpCAM, GAPDH, GJA1, GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, GUSB, KRT18, MAPK1, MAPK3, MME, Notch1, NOV1, PECAM, PRKACA, PRKACB, PRKACG, PRKC A subject is diagnosed with endometriosis if the expression of one or more genes from a group containing A, SNAI1, SRC, TGFBR2, TJAP1, TJP1, TJP2, Twist1, VEGFR1, VIM, Zeb2, ZO1, and ZO2 is elevated compared to the expression of one or more of the genes in endometrial epithelial cells from a subject without endometriosis, or is decreased compared to the expression of one or more of the genes in endometrial stromal cells from a subject without endometriosis.

[0054] The expression level of the above genes increases or decreases gradually with the severity of disease.Therefore, in one embodiment, the expression level of genes can be used to determine the stage of endometriosis.The genes that have the most differential expression between endometriosis epithelial cell samples and normal endometrial epithelial cell samples are in the example (Table 1 below): CDH2, vimentin and CTNNB.

[0055] (Table 1) Expression of various genes in endometrial epithelial cells Log 10 expression * : TIFF0007733931000001.tif66136172: Normal 164: Endometriosis Stage I / II 172: Endometriosis stage III / IV *Expression is 2 -ΔCt where Ct is the PCR cycle threshold for each gene, and Δ (Greek, delta) is the difference between the target gene and the normalized housekeeping gene (GAPDH).

[0056] In one embodiment, the woman being tested for endometriosis has symptoms of endometriosis or has a family history of the disease. The woman being tested for endometriosis may be undergoing infertility treatment or may have infertility. In one embodiment, the woman being tested for endometriosis is of reproductive age. The woman may be between 15 and 45 years old.

[0057] D. Endometriosis medication In one embodiment, the subject is diagnosed with endometriosis based on the expression level of the disclosed gene, and then undergoes treatment for endometriosis.The treatment for endometriosis is aimed at alleviating the symptoms of the disease.The most common symptoms of this disease include pain, bleeding and infertility.

[0058] i. Painkillers One embodiment provides a method for treating endometriosis-induced pain. Analgesics can be used for mild pain and inflammatory symptoms of endometriosis. The most common analgesics are nonsteroidal anti-inflammatory drugs (NSAIDs). Representative examples of nonsteroidal anti-inflammatory drugs include oxicams such as piroxicam, isoxicam, tenoxicam, and sudoxicam; salicylates such as aspirin, disalcid, benorylate, trilisate, safaprin, solprin, diflunisal, and fendosal; acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; mefenamic acid, meclofenamic acid, Nonsteroidal anti-inflammatory drugs include, but are not limited to, fenamic acids such as flufenamic acid, nifluric acid, and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethasone.Mixtures of these nonsteroidal anti-inflammatory drugs can also be used.

[0059] Steroidal anti-inflammatory agents can also be used to treat pain. Representative examples of steroidal anti-inflammatory agents include hydrocortisone, hydroxyl-triamcinolone, α-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoximetasone, deoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortisone, Corticosteroids include, but are not limited to, texolone, flucetonide, fludrocortisone, fluradrenolone, fludrocortisone, diflorasone diacetate, fluradrenolone acetonide, medrysone, amcinfel, amcinafide, betamethasone and its remaining esters, chloroprednisone, chloroprednisolone acetate, clocortolone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0060] If the pain is so severe that anti-inflammatory medications are ineffective, women may be prescribed narcotic pain medications such as, but not limited to, morphine, fentanyl, oxycodone, tramadol, hydromorphone, and codeine.

[0061] ii. Hormonal therapy drugs Another treatment for endometriosis-related pain and bleeding is hormonal therapy. Because ectopic endometrial tissue undergoes a cycle similar to menstruation, hormones may be effective in treating pain associated with the disease. Hormones can be delivered in the form of a pill, an injection or injectable, or a nasal spray. Oral contraceptives can be used to deliver hormones. Oral contraceptives typically contain two hormones: estrogen and progestin. Exemplary combination oral contraceptives include, but are not limited to, desogestrel / ethinyl estradiol, dienogest / estradiol valerate, drospirenone / ethinyl estradiol, drospirenone / ethinyl estradiol / levomefolate, ethynodiol / ethinyl estradiol, levonorgestrel / ethinyl estradiol, mestranol / norethindrone, norethindrone / ethinyl estradiol, norgestimate / ethinyl estradiol, and norgestrel / ethinyl estradiol.

[0062] Progestins are a group of drugs that act similarly to the female hormone progesterone. Progestins can be taken as a pill, by injection, or through an intrauterine device (IUD). The most commonly prescribed oral contraceptives are combination estrogen and progestin preparations, although progestin-only contraceptives are also prescribed. Oral contraceptives have been shown to improve the symptoms of endometriosis by reducing or completely stopping a woman's menstrual cycle.

[0063] Gonadotropin-releasing hormone (GnRH) agonist is generally prescribed to women with endometriosis.GnRH agonist comes in various forms, including injection every three months, injection every month, injection every day, and nasal spray.Exemplary GnRH agonist includes but is not limited to buserelin, goserelin, leuprorelin, leuprolide, nafarelin, and triptorelin.

[0064] Danazol is an androgen that has been shown to be effective in treating pelvic pain associated with endometriosis.

[0065] In one embodiment, a woman diagnosed with endometriosis using the disclosed methods is treated with a hormone therapy drug.

[0066] iii.Surgical treatment Surgery has been shown to provide significant pain relief from endometriosis. Laparoscopy is a type of surgery in which a surgeon makes a small incision in the abdomen and inserts a small viewing instrument called a laparoscope into the abdomen. This allows the surgeon to directly visualize endometriotic lesions. The surgeon may make a secondary incision to insert a laser or other instrument into the abdomen to remove or destroy the lesions. In one embodiment, patients diagnosed with endometriosis using the methods disclosed herein are treated by laparoscopy.

[0067] Laparotomy is a major abdominal surgery that can also be used to remove endometriotic lesions. In this procedure, the surgeon makes an incision across the abdomen to visualize the abdominal cavity and uterus. The surgeon can remove endometriotic lesions during the laparotomy. In one embodiment, a patient diagnosed with endometriosis using the methods disclosed herein is treated with a laparotomy. The surgeon can also perform a hysterectomy, in which the entire uterus is removed, during the laparotomy. Patients with extreme pain or advanced or recurrent endometriosis can choose to undergo a hysterectomy to eradicate endometriosis. In one embodiment, a patient diagnosed with endometriosis using the methods disclosed herein is treated with a hysterectomy.

[0068] Women with central abdominal pain may have their pelvic nerves severed to relieve the pain. This can be done via laparoscopy or laparotomy. There are two procedures that severe different nerve pathways. Presacral neurectomy severs the nerves that connect to the uterus. In one embodiment, patients diagnosed with endometriosis using the methods disclosed herein are treated with presacral neurectomy. A second procedure, called laparoscopic uterine nerve ablation, involves severing the nerves in the ligaments that anchor the uterus. In one embodiment, patients diagnosed with endometriosis using the methods disclosed herein are treated with laparoscopic uterine nerve ablation. [Example]

[0069] Example 1. Gap junction genes are elevated in endometriosis-derived uterine epithelial cells and decreased in endometriosis-derived stromal cells method Obtaining eutopic (uterus) endometrial tissue from women with and without endometriosis The presence or absence of endometriosis was confirmed by laparoscopy. All eutopic endometrial samples were obtained during the proliferative phase of the menstrual cycle from hormone-naive, normally cycling, reproductive-age women (age range: 30–45 years). Endometriosis samples were obtained from patients classified as stage I–IV according to the American Society of Reproductive Medicine classification. For normal subjects, endometrial tissue was isolated from women undergoing tubal sterilization who were not taking oral contraceptives, had a laparoscopy demonstrating the absence of endometriosis, and had no evidence of submucosal fibroids or endometrial polyps. Isolation of primary endometrial epithelial cells (EECs) and stromal cells (ESCs) from biopsy specimens was performed using a method developed by Kirk and Irwin, which has been shown to achieve approximately 97% purity, as confirmed by previous studies. Furthermore, the epithelial nature of EECs was confirmed in this study using staining for epithelial cell adhesion molecule (EpCAM) and cytokeratin 18 expression. ESCs were confirmed by vimentin staining. Table 2 shows a list of normal subjects and patients used in the study. Numbers were limited due to the extensive analysis performed on single cells and the need not extensively passage primary cells prior to analysis.

[0070] (Table 2) Patient base TIFF0007733931000002.tif73155

[0071] cell culture Primary endometrial EECs and ESCs were cultured in Dulbecco's modified Eagle's medium (DMEM) / F12 (1:1) (Sigma, St. Louis, MO, USA) containing antibiotics and antimycotics, 5 μg / mL insulin (Sigma), and 10% fetal bovine serum (Hyclone, Logan, UT, USA) as previously described. Experiments were performed using low passages (≤4). Established LP9 cells (Corriell Cell Repositories, Camden, NJ) were used as a model of peritoneal mesothelial cells. To examine connexin 43 (Cx43) protein expression, a standard immunofluorescence staining method was used on ESCs in culture using a green fluorescent-conjugated anti-Cx43 antibody. To experimentally suppress Cx43 expression in ESCs, cells were transfected with Cx43-specific siRNA.

[0072] Single-cell RNA screening of connexin gene panel expression by microfluidic PCR Primary EEC and ESC cultures were subjected to C1 (Fluidigm Corp) single-cell isolation and processing to generate amplified cDNA from each cell. The cDNA was then subjected to microfluidic PCR gene expression analysis using the Biomark platform (Fluidigm Corp). Corresponding PCR primer sequences for connexin and gap junction regulatory panels were used to detect the expression of these genes. In each microfluidic PCR chip assay, universal RNA (200 pg) derived from human normal tissue (catalog no. 4234565, BioChain, Newark, CA) and a no-template control (NTC) were used as positive and negative controls, respectively. Valid PCR products were determined by amplicon melting temperature curves for each gene amplicon.

[0073] result A pattern of decreased gap junction gene expression (top of each plot in Figures 1A-1I) was observed in endometrial stromal cells from endometriosis patients compared to those from healthy subjects, particularly in stage III / IV endometriosis disease (Figures 1A-1E). Importantly, this was independent of the menstrual phase from which the cells were collected.

[0074] In contrast, we observed a pattern of increased expression of many gap junction genes in endometriotic-enriched epithelial cells compared with normal samples (Figures 1F-1I). A gradual increase in gene expression is observed throughout the disease process, but for some genes, this increase was even pronounced in the earliest stages of endometriosis.

[0075] Changes were also observed in other genes involved in adhesive and sealed cell-cell contacts, as well as in the kinases that regulate them (Figures 1A-1I, bottom of each plot), but these were much less consistent compared to the connexin patterns described above.

[0076] Figures 2A-2BB show quantitative PCR analysis of all connexin expression at the single-cell level from normal subjects and early- and late-stage endometriosis patients after isolation of stromal (Figures 2A-2N) and epithelial cells (Figures 2O-2BB). Sample identifiers are listed in Table 3. Virtually all connexin genes showed a gradual decrease in expression with disease progression in stromal cells. Epithelial cells showed the opposite pattern, while the major connexin (GJA1) showed little difference between patients.

[0077] Table 3: Sample identifiers for Figures 2A-2BB and 3A-3N TIFF0007733931000003.tif25128

[0078] Figures 3A-3N show similar analyses of genes involved in regulating gap junction activity (PRKAC, PRKCB, Cav), or genes encoding cytoskeletal anchor proteins (Vim), or other types of cell-cell interactions, such as adherens junctions (CDH2, CTNNB) and tight junctions (TJP1). While some genes showed no change with disease (TJP1, Cav in epithelial cells, and others not shown), others showed expression patterns mimicking connexins (decreased with disease in stromal cells but increased in epithelial cells).

[0079] Example 2. Coupling and invasiveness of endometrial epithelial cells (E) and stromal cells (S) with mesothelial cells (M) method Homotypic and heterotypic gap junction intercellular communication (GJIC) assays (also described as coupling assays) GJIC was measured using the intercellular transfer of the fluorescent dye calcein, which can permeate through gap junctions. The assay was performed in culture medium supplemented with charcoal-stripped FBS (10%). Donor cells were incubated with calcein AM for 20 minutes at room temperature. Intracellularly, calcein AM was cleaved to calcein by nonspecific esterases, rendering it unable to diffuse through the cell membrane. Recipient cells were grown to confluence. Calcein-labeled donor cells were then dropped ("parachuted") onto the recipient cell layer, and calcein transfer between donor and recipient cells was observed by fluorescence microscopy imaging. For homotypic interactions, endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and mesothelial (LP9) donor cells were parachuted onto recipient cells of the same type, respectively. For heterotypic GJIC assays, EEC (or ESC) were parachuted onto LP9 recipient cells, and vice versa. Initial optimization assays showed that dye transfer in EEC, ESC, and LP9 cells was optimally observed 1.5–2 hours after parachute transfer. Fluorescent images of 10–15 fields per well were captured on an Operetta automated microscope (Perkin Elmer). A program written by Perkin Elmer allowed identification of all cells on the plate (from phase-contrast images), as well as the original donor (100 ± 50 per well), and dye-loaded recipients by calcein transfer over time. Data are expressed as the number of fluorescent recipient cells / number of donor cells for each condition.

[0080] Transmesothelial invasion assay A 3D invasion assay modeling transmesothelial invasion has been previously described. Briefly, LP9 peritoneal mesothelial cells (PMCs) were grown to confluence in 24-well invasion chamber inserts containing growth factor-reduced Matrigel™ coated on an 8-μm pore membrane (BD Bioscience, San Jose, CA, USA). 20,000 endometrial epithelial cells (EECs) or endometrial stromal cells (ESCs) were then labeled with CellTracker Green® (Molecular Probes-Invitrogen, Carlsbad, CA) and seeded on the confluent layer of LP9 PMCs in the prepared inserts and treated with the appropriate siRNA. After 20 hours of incubation, non-invaded cells on the top surface of the insert membrane were mechanically removed. Invaded cells on the bottom of the coated membrane were stained with DAPI and visualized using a fluorescent microscope with a 20x objective. Invasion assays for each cell type were performed in triplicate.

[0081] result Cell coupling was measured using a parachute assay, which measures calcein transfer over time between donor cells dropped onto a monolayer of recipient cells (Figures 4A-4F). This transfer rate was determined for homotypic coupling between either epithelial cells (EECs - Figure 4G) or stromal cells (ESCs - Figure 4H) in normal (N), early (I-II), and late (III-IV) endometriosis patients (black bars), as well as heterotypic coupling of these cells with LP9 peritoneal mesothelial cells (gray bars). Mesothelial cells induced coupling in patient-derived stromal cells, but not in normal subjects (Figure 4H), and in epithelial cells from either patients or normal subjects (Figure 4G).

[0082] Immunofluorescence staining for Cx43 reveals an internal distribution of Cx43 in stromal cells from all subjects (note that staining is concentrated around the cell edges and not at the cell-cell interface—Figure 5A–C). This is more pronounced as endometriosis progresses, although, surprisingly, there is no significant decrease in expression (Figure 5A–5C). However, exposure of stromal cells to mesothelial cells causes a redistribution of Cx43 to the cell surface (arrows) in endometriotic samples (Figure 5E and 5F), but to a much lesser extent in cells from normal subjects (Figure 5D). This activation of intracellular Cx43 stores upon exposure to mesothelial cells could explain the dramatic and rapid increase in heterotypic coupling seen in Figure 4B–4E.

[0083] Invasion of endometrial epithelial cells (E) and stromal cells (S) through mesothelial cell monolayers was also measured in Boyden chambers to mimic features of the invasive process in endometriosis (Figure 6). Endometrial cells were either left untreated (black bars) or treated with siRNA targeting Cx43 (gray bars) or the control protein GAPDH (hatched bars). Epithelial cells were invasive in a Cx43-dependent manner, which varied between patients and did not correlate with disease status (Figure 6). In contrast, stromal cells showed increased invasiveness with disease progression, which was solely dependent on Cx43 in the disease state.

[0084] In the foregoing specification, the invention has been described in relation to specific embodiments thereof, and while numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to further embodiments and that the specific details described herein may be varied considerably without departing from the underlying principles of the invention.

[0085] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics, and therefore, reference should be made to the appended claims, rather than the above specification, as indicating the scope of the invention.

Claims

1. 1. A method for detecting specific gene expression patterns in a cell population obtained from endometrial stromal cells of a subject, comprising: enriching or expanding endometrial stromal cells obtained from the subject; subjecting the enriched or expanded endometrial stromal cells to single cell processing in a microfluidic chamber to produce cDNA; subjecting the cDNA to PCR to detect RNA gene expression of one or more genes selected from the group consisting of GJA3, GJA5, GJA8, GJA9, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC2, and combinations thereof; wherein decreased expression of the one or more genes compared to expression of the one or more genes in endometrial stromal cells from a subject without endometriosis is an indication that the subject has endometriosis that is deep invasive endometriosis (stage III / IV).

2. 10. The method of claim 1, wherein the endometrial stromal cells are obtained from menstrual blood.

3. 10. The method of claim 1, wherein the endometrial stromal cells are obtained from an endometrial biopsy.

4. 10. The method of claim 1, further comprising staging the endometriosis.

5. The method of claim 1 , wherein the subject has symptoms of endometriosis.

6. 10. The method of claim 1, wherein the subject has previously been diagnosed with endometriosis.

7. 10. The method of claim 1, wherein the endometrial stromal cells are isolated by sorting the cells using endometrial stromal cell markers CD10, CD146, and CD13.

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