Improved methods for the detection and treatment of endometriosis

Abstract

Methods for treating and non-invasively detecting endometriosis using menstrual discharge are provided.

Claims

1. A method for providing information on endometriosis by analyzing collected menstrual discharge (ME) samples, The steps include (i) passing the sample through a 70 μm pore filter or (ii) a filter that allows ME single cells to pass through but not ME tissue fragments, so as to separate ME tissue fragments from ME single cells, The steps include collecting the aforementioned ME tissue fragments, The steps include processing the ME tissue piece so that it deaggregates into cells, The steps include (i) performing qPCR and / or digital droplet PCR gene expression analysis on the cells, (ii) single-cell RNA sequencing (scRNA-seq) analysis, (iii) flow cytometry, or (iv) protein expression analysis, (1) A step of determining the presence or absence of stromal cells or epithelial cells exhibiting a phenotype or gene expression pattern associated with endometriosis, based on the results of qPCR or digital droplet PCR gene expression, scRNA-seq analysis, flow cytometry or protein expression analysis, and / or (2) Based on the results of the qPCR gene expression or scRNA-seq analysis, flow cytometry or protein expression analysis, determine the levels of (a) uterine NK cells, (b) B cells and / or (c) T cells, and determine whether the levels of uterine NK cells, B cells and / or T cells are above, below, or within a predetermined control range for each of the levels of uterine NK cells, B cells and / or T cells, respectively. Equipped with, The presence of stromal cells exhibiting phenotypes, gene expression patterns, or protein expression patterns associated with endometriosis provides information that the sample is associated with endometriosis, and / or A method for indicating that levels of B cells and / or T cells above a predetermined control range, and levels of uterine NK cells below a predetermined control range, indicate that the sample is associated with endometriosis.

2. A method for identifying subjects who have dysmenorrhea and should be treated with progestin, progestin and estrogen, danazol, gonadotropin-releasing hormone agonist, aromatase inhibitor, or birth control pills, The procedure includes a step for identifying the subject's dysmenorrhea as indicating or not indicating endometriosis, the step for identification including the following method, the method being: The step of passing a sample of menstrual discharge (ME) from the subject through a 70 μm pore filter or a filter that allows ME single cells to pass through but not ME tissue fragments, in order to separate ME tissue fragments from ME single cells, The steps include collecting the aforementioned ME tissue fragments, The steps include processing the ME tissue piece so that it deaggregates into cells, The steps include (i) performing qPCR gene expression analysis, (ii) scRNA-seq analysis, (iii) flow cytometry, or (iv) mass spectrometry on the aforementioned cells, and thereafter, (1) A step of determining the presence or absence of stromal cells showing a phenotype or gene expression pattern associated with endometriosis based on the results of qPCR gene expression, scRNA-seq analysis, or flow cytometry, and / or (2) Based on the results of the qPCR gene expression or scRNA-seq analysis, determine the levels of (a) uterine NK cells, (b) B cells and / or (c) T cells, and determine whether the levels of uterine NK cells, B cells and / or T cells are above, below, or within a predetermined control range for each of the levels of uterine NK cells, B cells and / or T cells, respectively. Equipped with, The presence of stromal cells exhibiting a phenotype or gene expression pattern associated with endometriosis indicates that the sample is from a subject with dysmenorrhea that should be treated with progestin, progestin and estrogen, danazol, gonadotropin-releasing hormone agonist, aromatase inhibitor, or birth control pill, and / or A method for indicating that the level of B cells and / or T cells above a predetermined control range, and the level of uterine NK cells below a predetermined control range, is from a subject having dysmenorrhea that should be treated with progestin, progestin and estrogen, danazol, gonadotropin-releasing hormone agonist, aromatase inhibitor, or birth control pill.

3. The method according to claim 2, further comprising the step of enriching the sample with respect to stromal cells by removing CD45+ cells from the sample before performing (i) qPCR gene expression analysis, (ii) scRNA-seq analysis, (iii) flow cytometry, or (iv) mass spectrometry.

4. The method according to claim 2 or 3, further comprising the step of depleting epithelial cells from the sample before (i) qPCR gene expression analysis, (ii) scRNA-seq analysis, (iii) flow cytometry, or (iv) mass spectrometry.

5. The method according to any one of claims 2 to 4, wherein the step of treating the ME tissue piece to deaggregate the tissue piece into cells comprises the step of contacting the ME tissue piece with collagenase, DNase and / or liberase.

6. The method according to any one of claims 2 to 5, further comprising the step of freezing and / or storing the cells in a preservative or RNA stabilizing solution before or after deaggregating the tissue fragment.

7. Before performing (i) qPCR gene expression analysis, (ii) scRNA-seq analysis, (iii) flow cytometry, or (iv) mass spectrometry, A step of lysing the red blood cells in the aforementioned sample, The steps of depleting neutrophils from the aforementioned sample, Step of removing dead cells from the aforementioned sample. The method according to any one of claims 2 to 6, further comprising one or more of the above.

8. The method according to any one of claims 2 to 7, further comprising the step of passing the ME tissue fragments isolated from the ME single cells through a second filter having a pore size of 40 μm, wherein the step of collecting the ME tissue fragments is performed on the ME tissue fragments that do not pass through the second filter.

9. The method according to any one of claims 2 to 8, wherein the sample is collected in a menstrual cup or menstrual sponge.

10. The method according to any one of claims 2 to 9, further comprising the step of separating the stromal cells, uterine NK cells, B cells and / or T cells from each other using a surface marker before performing (i) qPCR gene expression analysis, (ii) scRNA-seq analysis, (iii) flow cytometry or (iv) mass spectrometry on the cells.

11. The method according to claim 10, wherein separation and / or isolation are performed using fluorescence-activated cell sorting or magnetically activated cell sorting.

12. The method according to any one of claims 1 to 11, further comprising the step of determining the level of stromal cells based on the results of qPCR gene expression or scRNA-seq analysis.

13. The method according to any one of claims 2 to 12, comprising the step of determining the presence or absence of stromal cells exhibiting a phenotype or gene expression pattern associated with endometriosis based on the results of the qPCR gene expression or scRNA-seq analysis, but without determining the levels of (a) uterine NK cells, (b) B cells and / or (c) T cells.

14. The method according to any one of claims 2 to 12, further comprising the step of determining the levels of (a) uterine NK cells, (b) B cells and / or (c) T cells based on the results of the qPCR gene expression or scRNA-seq analysis, and determining whether the levels of uterine NK cells, B cells and / or T cells are above, below, or within a predetermined control range for each of the levels of uterine NK cells, B cells and / or T cells.

15. The method according to any one of claims 2 to 14, wherein the subject is a human.

16. The method according to claim 15, wherein the subject is a young adult.

17. The method according to any one of claims 2 to 12 or 14 to 16, wherein the predetermined control range for B cells, T cells and / or uterine NK cells is determined from one or more ME tissue samples from one or more control subjects that do not have endometriosis.

18. A method for preparing a sample of menstrual discharge (ME) for analysis, such that the interstitial cell content in the sample is concentrated from 3% or less to 10% or more, The steps of passing the sample of menstrual discharge (ME) through (i) a 70 μm pore filter or (ii) a filter that allows ME single cells to pass through but not ME tissue fragments, in order to separate ME tissue fragments from ME single cells, A step of collecting ME tissue fragments that did not pass through the filter, The step of enzymatically treating the ME tissue piece so that the tissue piece deaggregates into cells, The steps include fixing, permeabilizing, and / or freezing the cells in methanol and / or other preservatives or RNA stabilizing solutions before or after deaggregation of the tissue fragments, Equipped with, A method for obtaining a stromal cell content of more than 10% in the sample by the above preparation.

19. The method according to claim 18, further comprising the step of preparing an ME sample for analysis so as to concentrate the stromal cell content in the aforementioned sample to 20% or more.

20. The method according to any one of claims 2 to 19, wherein the stromal cells are stromal fibroblasts (SFCs).

21. The method according to any one of claims 2 to 19, wherein the stromal cells are human CD45- / CD326- / CD31- / CD90+ / CD105+ / CD73+.

22. The method according to claim 21, wherein the stromal cells are CD140b+.

23. The method according to any one of claims 2 to 22, wherein the level of the uterine NK cells is determined and the uterine NK cells are proliferative uterine NK cells.

24. The method of claim 23, wherein the proliferative uterine NK cells are positive for a human marker of proliferative Ki-67 protein.

25. The method according to any one of claims 2 to 24, wherein the level of proliferative uterine NK cells in an endometriosis subject sample is at least four times lower than that in a control sample from a non-endometriosis subject.

26. The method according to any one of claims 2 to 25, wherein the level of proliferative uterine NK cells in an endometriosis subject sample is at least 10 times lower than that in a control sample from a non-endometriosis subject.

27. The method according to any one of claims 23 to 26, further comprising the step of selecting proliferative uterine NK cells based on the expression of cell proliferation-related markers.

28. The method according to claim 27, wherein the cell proliferation-related marker is a human marker of proliferative Ki-67, CENPF, UBE2C, ASPM, TOP2A, CKS1B, PCLAF, or NUSAP1.

29. The method according to claim 27 or 28, wherein the cells other than proliferative uterine NK cells are depleted from the sample by a method comprising negative antibody selection.

30. The step of determining the level of uterine NK cells is based on the results of the qPCR gene expression or scRNA-seq analysis, The method according to any one of claims 2 to 29, further comprising the step of determining whether the expression level of proliferative uterine NK cell human MKI67 or other cell proliferation-related markers is above, below, or within a predetermined control range for each of the proliferative uterine NK cell human MKI67 or other cell proliferation-related markers.

31. The method according to any one of claims 2 to 22, wherein the presence or absence of stromal cells exhibiting a phenotype or gene expression pattern associated with endometriosis is determined.

32. The method according to claim 31, wherein the presence of stromal cells in the sample exhibiting higher levels of human matrix gla protein (MGP), interleukin 11 (IL11), or insulin-like growth factor-binding protein 1 (IGFBP1) than the control indicates a phenotype or gene expression pattern associated with endometriosis.

33. The method according to any one of claims 2 to 32, wherein the stromal cells exhibit a phenotype or gene expression pattern associated with endometriosis, and the phenotype or gene expression pattern is pro-inflammatory or senescent.

34. A diagnostic kit for non-invasively diagnosing endometriosis in a subject, comprising: (A) a sample of menstrual discharge (ME); (B) (i) a 70 μm pore filter or (ii) a filter that allows ME single cells to pass through but not ME tissue fragments; and (C) an amount of collagenase, DNase and / or liberase effective for deaggregating a predetermined amount of ME tissue fragments before or after fixation.

35. The kit according to claim 34, further comprising a predetermined amount of preservative and / or RNA stabilizing solution.

36. A diagnostic kit for non-invasively diagnosing endometriosis in a subject, comprising: (A) (i) a 70 μm pore filter or (ii) a filter that allows ME single cells to pass through but not ME tissue fragments; and (B) an amount of collagenase, DNase and / or liberase effective for deaggregating a predetermined amount of ME tissue fragments before or after fixation.

37. The kit according to claim 36, further comprising a predetermined amount of preservative and / or RNA stabilizing solution.

Images