BACKGROUND OF THE INVENTION
The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Endometriosis is a chronic and underdiagnosed disease which affects 5-10% of women of childbearing age and is characterized by growth of endometrial-like tissue outside of the uterus, most often in the peritoneal cavity. Delay in diagnosis is a major problem for management of this disorder, and treatment is often not initiated until the disease has progressed for many years. Currently in the US, diagnosis of endometriosis can take up to 7 to 10 years for an individual. Endometriosis is a very complex disease, sometimes with vague and non-specific symptoms. Many women present, for example, with GI symptoms and they end up seeing a gastroenterologist as part of their diagnostic journey. Thus, it is critically important to find methods and means for accurate and early diagnosis of endometriosis. In addition, dysmenorrhea in adolescents has both endometriosis-related and endometriosis-unrelated occurrences. To avoid unnecessary or incorrect intervention in this population, it is important to be able to distinguish between those who would benefit from endometriosis treatment and those for whom such treatment will be of no benefit or a burden.
Although the exact etiology of endometriosis remains unknown, retrograde menstruation is recognized as a common underlying factor leading to the deposit of menstrual effluent (ME) into the peritoneal cavity. Differences in the cellular biology and genetics of the cells within ME are therefore likely to explain why endometriosis develops in only a subset of women. In addition, invasive diagnostic techniques are not preferred by patients.
The present invention addresses these needs and provides methods of noninvasively detecting and treating endometriosis.
SUMMARY OF THE INVENTION
A method of non-invasively diagnosing endometriosis in a subject comprising:
- passing a sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments (i.e. shed endometrial tissue), so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments; treating the ME tissue fragments so as to disaggregate the tissue fragments into cells; performing (i) qPCR and/or digital droplet PCR gene expression analysis or (ii) single cell RNA-sequencing (scRNA-seq) analysis on the cells or (iii) flow cytometry or (iv) other protein analysis; then (1) determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis, or protein expression by flow cytometry, and/or
- (2) determining levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry or other protein analysis and determining if the uterine NK cell, B cell, T cell levels are above, below, or within a predetermined control range for uterine NK cell, B cell, T cell levels, respectively; wherein the presence of stromal cells exhibiting a phenotype, or gene or protein expression pattern associated with endometriosis indicates that the sample is from a subject having endometriosis, and/or
- a B cell and/or T cell level above the predetermined control range, and a uterine NK cell level below the predetermined control range, indicates that the sample is from a subject having endometriosis.
A method of treating a subject with a dysmenorrhea for endometriosis comprising:
- (A) obtaining an identification of the dysmenorrhea in the subject (a) as indicative of endometriosis or (b) as not indicative of endometriosis, wherein identification has been determined by a method comprising:
- passing a sample of menstrual effluent (ME) from the subject through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments;
- treating the ME tissue fragments with enzymes so as to disaggregate the tissue fragments into cells, red blood cell lysis and neutrophil depletion;
- performing (i) qPCR or digital droplet PCR for gene expression analysis or (ii) scRNA-seq analysis or (iii) or flow cytometry, (iv) or other protein expression analysis on the cells; then
- (1) determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis, or protein expression by flow cytometry, and/or
- (2) determining levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometery or other protein analysis and determining if the uterine NK cell, B cell, T cell levels are above, below, or within a predetermined control range for uterine NK cell, B cell, T cell levels respectively; wherein the presence of stromal cells exhibiting a phenotype, or protein or gene expression pattern, associated with endometriosis indicates that the sample is from a subject having dysmenorrhea indicative of endometriosis, and/or
- a B cell and/or T cell level above the predetermined control range, and a uterine NK cell level below the predetermined control range indicates that the sample is from a subject having dysmenorrhea indicative of endometriosis;
- and
- (B) treating the subject who has been identified as having a dysmenorrhea indicative of endometriosis with an amount of a progestin, a progestin and an estrogen, a danazol, a gonadotropin-releasing hormone agonist, or a birth control pill to the subject effective to treat endometriosis.
A method of determining the efficacy of a treatment for endometriosis comprising:
- assessing a baseline level in menstrual effluent of a subject having endometriosis of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis, and/or levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or protein expression analysis by any of the methods disclosed herein;
- treating the subject by performing a laparoscopic surgery or hysterectomy on the subject, or administering an amount of a progestin, a progestin and an estrogen, a danazol, a gonadotropin-releasing hormone agonist, or a birth control pill to the subject effective to treat endometriosis;
- assessing a post-treatment level in menstrual effluent from the subject of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis, and/or levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq or protein expression analysis;
- comparing the post-treatment level with the baseline level of the subject, wherein an improvement in levels of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis, and/or an improvement in levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells indicates that the treatment is efficacious.
A method of preparing a menstrual effluent (ME) sample for analysis so as to enrich stromal cell content in the sample from 1%, or less, to 10%, or over, comprising: passing the sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting ME tissue fragments that have not passed through the filter;
- enzymatically treating fresh or fixed ME tissue fragments so as to disaggregate the tissue fragments into cells; and freezing the cells in a preservative (e.g., methanol or formaldehyde) prior to and/or subsequent to disaggregating the tissue fragments, wherein the preparation results in a stromal cell content in the sample of over 10%.
A kit for non-invasively diagnosing endometriosis in a subject comprising a menstrual effluent (ME) sample; (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments; an amount of a collagenase and/or DNase and/or liberase effective to disaggregate an amount of ME tissue fragments.
A method of treating endometriosis in a subject comprising obtaining an identification of the subject as in need of treatment of endometriosis, wherein the subject has been identified as having endometriosis by any of the methods disclosed herein, and treating the subject by performing a laparoscopic surgery or hysterectomy on the subject, or administering an amount of a progestin, a progestin and an estrogen, a danazol, a gonadotropin-releasing hormone agonist, or a birth control pill to the subject effective to treat endometriosis.
Also provided is a method of identifying patients at risk for endometriosis by identifying subclinical inflammation of the uterine lining (e.g., acute or chronic endometritis) by a method comprising passing a sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments; using fresh or fixed tissue fragments;
- treating the ME tissue fragments so as to disaggregate the tissue fragments into cells; performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis or (ii) protein expression analysis on the cells;
- then
- (1) determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with subclinical inflammation of the uterine lining based on results of the qPCR gene expression or scRNA-seq analysis or protein expression analysis, and/or
- (2) determining levels of (a) uterine NK cells, (b) B cells, (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis and determining if the uterine NK cell, B cell, and/or T cell levels are above, below, or within a predetermined control range for uterine NK cell, B cell, and/or T cell levels respectively;
- wherein the presence of stromal cells exhibiting a phenotype, or gene expression pattern, associated with subclinical inflammation of the uterine lining indicates that the sample is from a subject having endometriosis, and/or
- a B cell and/or T cell level above the predetermined control range, and a uterine NK cell level below the predetermined control range indicates that the sample is from a subject having endometriosis.
DETAILED DESCRIPTION OF THE INVENTION
A method of non-invasively diagnosing endometriosis in a subject comprising: passing a sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments;
- treating the ME tissue fragments so as to disaggregate the tissue fragments into cells;
- performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis or (iii) protein expression analysis on the cells;
- then
- (1) determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis or protein expression analysis, and/or
- (2) determining levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or protein expression analysis and determining if the uterine NK cell, B cell, and/or T cell levels are above, below, or within a predetermined control range for uterine NK cell, B cell, and/or T cell levels respectively;
- wherein the presence of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis indicates that the sample is from a subject having endometriosis, and/or
- a B cell and/or T cell level above the predetermined control range, and a uterine NK cell level below the predetermined control range indicates that the sample is from a subject having endometriosis.
In the methods disclosed herein, after collecting the ME tissue fragments, they can be used fresh in subsequent steps or fixed first then used in subsequent steps.
A method of treating a subject with a dysmenorrhea for endometriosis comprising:
- (A) obtaining an identification of the dysmenorrhea in the subject (a) as indicative of endometriosis or (b) as not indicative of endometriosis, wherein identification has been determined by a method comprising:
- passing a sample of menstrual effluent (ME) from the subject through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments;
- treating the ME tissue fragments so as to disaggregate the tissue fragments into cells;
- performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis on the cells, or (iii) flow cytometry or (iv) other protein expression analysis on the cells; then
- (1) determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry or other protein expression analysis, and/or
- (2) determining levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry or other protein expression analysis and determining if the uterine NK cell, B cell, and/or T cell levels are above, below, or within a predetermined control range for uterine NK cell, B cell, and/or T cell levels respectively;
- wherein the presence of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis indicates that the sample is from a subject having dysmenorrhea indicative of endometriosis, and/or
- a B cell and/or T cell level above the predetermined control range, and a uterine NK cell level below the predetermined control range indicates that the sample is from a subject having dysmenorrhea indicative of endometriosis;
- and
- (B) treating the subject who has been identified as having a dysmenorrhea indicative of endometriosis with an amount of a progestin, a progestin and an estrogen, a danazol, a gonadotropin-releasing hormone agonist, an aromatase inhibitor, or a birth control pill to the subject effective to treat endometriosis.
A method of determining the efficacy of a treatment for endometriosis comprising:
- assessing a baseline level in menstrual effluent of a subject having endometriosis of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis, and/or levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry or other protein expression analysis by the method described herein;
- treating the subject by performing a laparoscopic surgery or hysterectomy on the subject, or administering an amount of a progestin, a progestin and an estrogen, a danazol, a gonadotropin-releasing hormone agonist, an aromatase inhibitor, or a birth control pill to the subject effective to treat endometriosis;
- assessing a post-treatment level in menstrual effluent from the subject of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis, and/or levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry or other protein expression analysis;
- comparing the post-treatment level with the baseline level of the subject, wherein an improvement in levels of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis, and/or an improvement in levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells indicates that the treatment is efficacious. In embodiments, a subject who has been identified as having a dysmenorrhea not indicative of endometriosis is treated for the dysmenorrhea with an amount of a nonsteroidal anti-inflammatory drug. In embodiments, the method further comprises one or more additional iterations of the method so as to determine when treatment can be stopped, wherein when no further improvement is seen in post-treatment levels, then treatment is stopped.
In embodiments, the uterine NK cell, B cell, and/or T cell levels are measured as a fraction or proportion of the relevant cell type as total cells in a sample. In embodiments, the uterine NK cell, B cell, and/or T cell levels are measured as a fraction or proportion of the relevant cell type as total cells in a sample and compared to the respective fraction or proportion of the same cell type in a control sample (e.g. from an otherwise equivalent but non-endometriosis sample.
In embodiments, the methods further comprise enriching the sample for stromal cells by removing CD45+ cells from the sample prior to performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis. In embodiments, the methods further comprise enriching the sample for stromal cells by removing CD45+ cells from the sample prior to performing flow cytometry or other protein expression analysis.
In embodiments, a subject who has been identified as having a dysmenorrhea not indicative of endometriosis is treated for the dysmenorrhea with an amount of a nonsteroidal anti-inflammatory drug. In embodiments, a subject is identified as having a dysmenorrhea not indicative of endometriosis by (a) having or being diagnosed with a dysmenorrhea but (b) not showing (1) the presence of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis, or (2) showing uterine NK cell, B cell, and/or T cell levels within a predetermined control range for uterine NK cell, B cell, and/or T cell levels respectively which predetermined control range is not associated with the presence of endometriosis.
In embodiments, the methods further comprise enriching the sample for stromal cells by removing CD45+ cells from the sample prior to performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis, or (iii) protein analysis
In embodiments, the methods further comprise depleting epithelial cells from the sample prior to performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis. In embodiments, epithelial cells are removed by a short adhesion step or by using depletion with anti-CD325/EpCAM. In embodiments, the methods further comprise isolating epithelial cells from the sample prior to performing (i) qPCR gene expression analysis, (ii) scRNA-seq analysis or (iii) protein analysis In embodiments, the methods further comprise depleting epithelial cells from the sample prior to performing flow cytometry or other protein expression analysis. flow cytometry or other protein expression analysis.
In embodiments, treating the ME tissue fragments is effected with enzymes so as to disaggregate the tissue fragments into cells. In embodiments, treating the ME tissue fragments further comprises one or more of red blood cell lysis and removing granulocytes. In embodiments, removing granulocytes is effected by CD66b selection, for example via a CD66b cocktail.
In embodiments, the methods can comprise performing (i) qPCR and/or digital droplet PCR gene expression analysis or (ii) single cell RNA-sequencing (scRNA-seq) analysis or (iii) flow cytometry, or (ii) protein expression analysis on the cells.
In embodiments, the method is performed on epithelial cells, myeloid cells, plasma cells, or eosinophils digested from tissue in the ME, mutatis mutandis.
In embodiments, treating the ME tissue fragments so as to disaggregate the tissue fragments into cells comprises contacting the ME tissue fragments with a collagenase. In embodiments, the collagenase is a collagenase I. In further embodiments, the tissue fragments are treated with a DNase and/or liberase. In embodiments, a collagenase I (1 mg/ml)/DNase (0.5 mg/ml)/liberase mixture is used on the tissue for 15 min/37° C. In embodiments, the resultant released cells may be subjected to RBC lysis, neutrophil depletion and/or Ficoll centrifugation to remove dead cells and/or positive immunoselection for specific cell types (stromal cells, T cells, uNK cells, epithelial cells) using antibody coated-magnetic beads before freezing in methanol or other preservative such as formaldehyde, for scRNA seq analysis, qPCR, and/or protein analysis
In embodiments, the methods further comprise freezing the cells using a preservative subsequent or prior to disaggregating the tissue fragments. In embodiments, the cryopreservative comprises methanol or formaldehyde. In embodiments, the methods further comprise freezing the cells in an RNA-stabilizing solution prior to or subsequent to disaggregating the tissue fragments
In embodiments, the methods can further comprise one or more of:
- lysing red blood cells in the sample;
- depleting neutrophils from the sample; and
- removing dead cells from the sample;
- prior to performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis or (iii) protein expression analysis.
In embodiments, the methods further comprise passing the ME tissue fragments separated from the ME single cells through a second filter having a 40 μm pore diameter and wherein the collecting the ME tissue fragments is performed on the ME tissue fragments that do not pass through the second filter.
In embodiments, the sample has been collected in a menstrual cup or a menstrual sponge. In embodiments, the ME sample has previously been collected from a subject.
In embodiments, the methods further comprise separating the stromal, uterine NK cells, B cells, and/or T cells, or tissue-derived epithelial cells, myeloid cells, plasma cells, eosinophils, or other cell types from one another using surface markers prior to performing (i) qPCR or digital droplet PCR gene expression analysis or (ii) scRNA-seq analysis on the cells or (iii) flow cytometry or (iv) other protein expression analysis.
In embodiments, separation is effected using fluorescence-activated cell sorting or magnetic-activated cell sorting. In embodiments, isolation is effected using fluorescence-activated cell sorting or magnetic-activated cell sorting.
In embodiments, the methods comprise determining levels of stromal cells based on results of the qPCR gene expression or scRNA-seq analysis. In embodiments, the methods comprise determining levels of stromal cells based on results of digital droplet PCR gene expression or scRNA-seq analysis or protein expression analysis.
In embodiments, the methods comprise determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis, but not determining levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells. In embodiments, the methods comprise determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, or protein expression pattern, associated with endometriosis based on results of the qPCR gene expression or scRNA-seq analysis, but not determining levels of epithelial cells, myeloid cells, plasma cells, or eosinophils digested from tissue in the ME In embodiments, the methods comprise determining levels of (a) uterine NK cells, (b) B cells, and/or (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis and determining if the uterine NK cell, B cell, and/or T cell levels (or other tissue-derived cell types) are above, below, or within a predetermined control range for uterine NK cell, B cell, and/or T cell levels (or other tissue-derived cell types) respectively.
In embodiments, the subject is a human. In embodiments the adult subject is pre-menopausal. In embodiments, the subject is an adolescent. In embodiments, the adolescent is 12 years to <18 years.
In embodiments of the methods, stromal cells are not cultured or maintained in culture prior to digestion and processing. In embodiments, the cells are not cultured prior to analysis. In embodiments, no enzymatic digestion of tissue samples occurs until after filtering to remove single cells.
In embodiments relating to endometriosis, the predetermined control range for B cells, T cells, uterine NK cells, and/or other tissue-derived cell types is determined from one or more ME tissue fragments from one or more control subjects who do not have endometriosis. In embodiments relating to acute or chronic endometritis, the predetermined control range for B cells, T cells and/or uterine NK cells (or other tissue-derived cell types) is determined from one or more ME tissue fragments from one or more control subjects who do not have chronic endometritis.
In embodiments the genes, proteins and/or nucleic acids referred to herein are human.
A method of preparing a menstrual effluent (ME) sample for analysis so as to enrich stromal cell content in the sample from 1%, or less, to 10%, or over, comprising: passing the sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting ME tissue fragments that have not passed through the filter;
- enzymatically treating fresh or fixed ME tissue fragments so as to disaggregate the tissue fragments into cells; and
- freezing the cells in a preservative (e.g., methanol or formaldehyde) prior to or subsequent to disaggregating the tissue fragments,
- wherein the preparation results in a stromal cell content in the sample of over 10%. In embodiments, the method further comprises preparing an ME sample for analysis so as to enrich the stromal cell content in the sample to 20% or more.
In embodiments, the method further comprises preparing an ME sample for analysis so as to enrich the stromal cell content in the sample to 20% or more.
In embodiments, the methods further comprise obtaining the ME sample from the subject.
In embodiments of the methods the stromal cells are stromal fibroblast cells (SFC). In embodiments of the methods the stromal cells are CD45−/CD326−/CD31−/CD 90+/CD105+/CD73+. In embodiments of the methods the stromal cells are CD140b+. In embodiments of the methods the stromal cells exhibit a phenotype, or gene expression pattern, associated with endometriosis, wherein the phenotype or gene expression pattern is a pro-inflammatory or a senescent phenotype or gene expression pattern.
In embodiments, the level of uterine NK cells is determined, and the uterine NK cells are proliferative uterine NK cells.
In embodiments, the proliferative uterine NK cells are positive for human marker of proliferation Ki-67 protein (encoded by MKI67).
In embodiments, the level of proliferative uterine NK cells in an endometriosis subject sample is at least 4-fold lower than in a control sample from a non-endometriosis subject.
In embodiments, the level of proliferative uterine NK cells in an endometriosis subject sample is at least 10-fold lower than in a control sample from a non-endometriosis subject.
In embodiments, the methods further comprise selecting for proliferative uterine NK cells based on expression of a cell proliferation-associated marker. In embodiments, the cell proliferation-associated marker is human marker of proliferation Ki-67, CENPF, UBE2C, ASPM, TOP2A, CKS1B, PCLAF or NUSAP1. In embodiments, the cells other than proliferative uterine NK cells are depleted from the sample by a method comprising negative antibody selection.
In embodiments, determining levels of uterine NK cells is based on results of the qPCR gene expression or scRNA-seq analysis and determining if the expression level of proliferative uterine NK cell human MKI67, or other cell proliferation-associated marker, is above, below, or within a predetermined control range for proliferative uterine NK cell human MKI67, or other cell proliferation-associated marker, respectively.
In embodiments, the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with endometriosis is determined.
In embodiments, the presence of stromal cells in the sample demonstrating higher level of human matrix Gla protein (MGP), interleukin 11 (IL11) or insulin like growth factor binding protein 1 (IGFBP1) than in a control is indicative of a phenotype, or gene expression pattern, associated with endometriosis.
A kit for non-invasively diagnosing endometriosis in a subject comprising a menstrual effluent (ME) sample; (i) a 70 mm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments; an amount of a collagenase and/or DNase and/or liberase effective to disaggregate an amount of ME tissue fragments. In embodiments, the kit further comprises an amount of preservative. In embodiments, the kit further comprises an amount of an RNA-stabilizing solution. In embodiments, the preservative comprises methanol or formaldehyde
A method of treating endometriosis in a subject comprising obtaining an identification of the subject as in need of treatment of endometriosis, wherein the subject has been identified as having endometriosis by any of the methods of disclosed herein, and treating the subject by performing a laparoscopic surgery t to remove endometriosis lesions, or administering an amount of a progestin, a progestin and an estrogen, a danazol, a gonadotropin-releasing hormone agonist, an aromatase inhibitor, or a birth control pill to the subject in an amount that is effective to treat endometriosis. In embodiments, treatment results in a reduction in one or more of the following symptoms in the subject; chronic pelvic pain, dysmenorrhea, dyspareunia, dysuria, dyschezia, bloating.
In embodiments the laparoscopic surgery is performed to remove ectopic lesions.
Also provided is a method of identifying patients at risk for endometriosis by identifying subclinical inflammation of the uterine lining (e.g. acute or chronic endometritis) by a method comprising passing a sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments; using fresh or fixed tissue fragments; treating the ME tissue fragments so as to disaggregate the tissue fragments into cells; performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis on the cells or (iii) flow cytometry or (iv) mass spectrometry or other protein analysis on the cells;
- then
- (1) determining the presence or not of stromal cells exhibiting a phenotype, or gene expression pattern, associated with subclinical inflammation of the uterine lining based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry or other protein expression analysis, and/or
- (2) determining levels of (a) uterine NK cells, (b) B cells, (c) T cells based on results of the qPCR gene expression or scRNA-seq analysis and determining if the uterine NK cell, B cell, and/or T cell levels are above, below, or within a predetermined control range for uterine NK cell, B cell, and/or T cell levels, respectively; wherein the presence of stromal cells exhibiting a phenotype, or gene expression pattern associated with subclinical inflammation of the uterine lining indicates that the sample is from a subject having endometriosis, and/or a B cell and/or T cell level above the predetermined control range, and a uterine NK cell level below the predetermined control range indicates that the sample is from a subject having endometriosis.
The methods herein can be used to identify subjects as at risk for endometriosis. Also provided are methods or treating subjects at risk for endometriosis by administering to the subject locally or systemically an anti-inflammatory agent that targets a cytokine. In embodiments, the cytokines are TNFalpha and/or IL 1beta. Anti-inflammatory agents directed to cytokine(s) are known in the art, including certain organic small molecules (see, e.g. world wide web at ncbi.nlm.nih.gov/pmc/articles/PMC3752337/, incorporated by reference); anticytokines may also be biologics, e.g., monoclonal antibodies or fusion proteins directed against a known cytokine such as TNFalpha or IL-1beta).
Menstrual cups as described herein include, but are not limited to, those sold by Diva International Inc., Ontario, Canada. Sponges for collecting ME as discussed herein include, but are not limited to, polyether polyurethane menstrual sponges
A method of non-invasively diagnosing endometriosis in a subject comprising: passing a sample of menstrual effluent (ME) through (i) a 70 μm pore filter or (ii) a filter that permits through passage of ME single cells but not of ME tissue fragments, so as to separate ME tissue fragments from ME single cells;
- collecting the ME tissue fragments; using fresh or fixed tissue fragments;
- treating the ME tissue fragments so as to disaggregate the tissue fragments into cells;
- performing (i) qPCR gene expression analysis or (ii) scRNA-seq analysis on the cells or (iii) flow cytometry; or (iii) mass spectrometry or other protein analysis on the cells, then determining, based on results of the qPCR gene expression or scRNA-seq analysis or flow cytometry, if the (a) uterine NK cells, (b) B cells, and/or (c) T cells exhibit a gene expression pattern associated with endometriosis,
- wherein a gene expression pattern in (a) uterine NK cells, (b) B cells, and/or (c) T cells associated with endometriosis indicates that the sample is from a subject having endometriosis.
As used herein, a predetermined control amount is a value decided or obtained, usually beforehand, as a control. The concept of a control is well-established in the field, and can be determined, in a non-limiting example, empirically from non-afflicted subjects (versus afflicted subjects, including afflicted subjects having different grades of the relevant affliction), and may be normalized as desired (in non-limiting examples, for volume, mass, age, location, gender) to negate the effect of one or more variables.
“And/or” as used herein, for example with option A and/or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.
All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Experimental Details
The inventors have developed new methods of isolating and studying shed eutopic endometrial tissues with abundant stromal cells using ME. Also disclosed are new collection methods of fresh ME that that are practical for both adults and adolescents and can be repeated across menstrual cycles. Importantly, the methods capture the phenotypic state of the eutopic endometrium at a time when ME tissues are delivered into the peritoneal cavity. In addition, disclosed are innovations to rapidly digest endometrial tissue fragments in ME followed by or preceded by, fixation, permeabilization, and/or freezing in a preservative, e.g., in methanol or formaldehyde, so that scRNA-Seq or qPCR or protein analysis (or other sequence analysis methods) can be conveniently and cost-effectively “batched’ and carried out on multiple samples and analyzed simultaneously using, e.g., Demuxlet. This innovation also permits the application of these analyses to compare large numbers of patients and control ME samples, in addition to repeated ME sampling over time to ensure reproducibility. The results provide a full analysis of numerous ME cell types and individual cell subsets, including fresh stromal cells, epithelial cells, uterine NK cells, T cells, B cells, and other cells of the immune system. Interestingly, differences with cultured samples are quite apparent, as described hereinbelow.
Study subjects: Patient recruitment/enrollment has been through the ROSE study which has >1500 participants.
Decidualization defects in ME-eSCs: It has previously been shown that a decidualization defect associated with endometriosis can be readily observed in cultured endometrial stromal cells derived from menstrual effluent (ME-eSCs) (14, 16, 17). In addition, subjects with symptoms of endometriosis without a confirmed diagnosis have a similar defect. Prominent decidualization defects were seen in ME-eSCs (p1) from endometriosis subjects as reflected in production of IGFBP1 by ELISA, comparing cAMP to vehicle after 24 hrs (IGFBP1 ratio). Up to 10% of control subjects have relatively low decidualization capacity). Despite the high accuracy of this observational correlation this approach presents many challenges for developing a diagnostic, since ME-eSCs constitute only 1-3% of the free separated mononuclear cells in fresh ME, and these assays require culturing of these cells. While this is not difficult, but it is very time consuming and expensive, and therefore has limited use as a diagnostic test.
Endometrial tissues are abundant in ME: In the course of processing many samples of ME collected using a menstrual cup, it was observed that clumps of endometrial tissues are present that were not captured by initial approaches. These can be demonstrated by filtering the ME over, e.g., 70 μm filters to enrich for such fragments. Carrying this out on multiple samples it was discovered that intact endometrial tissues can be readily demonstrated by histological analysis of ME. Micrographs of samples showed the presence of fragments of endometrial tissues containing both epithelial and stromal cells, as well as uNK cells. Immunostaining confirmed the presence of endometrial stromal cells (CD10) and uNK cells (CD56) in five individuals.
Several approaches were explored to processing of ‘whole ME’ containing these tissue fragments for scRNA-Seq analysis. Initially, the entire ME sample was digested with collagenase and DNase to release cells from tissue fragments, followed by filtering to capture cells released by digestion, as well the free single cells in the ME. Neutrophils were depleted using magnetic meads and used as a source of genomic DNA for DEMUXLET analysis (neutrophils dominate the cellular content of single cells in ME). This was followed by density gradient separation using Ficoll to remove dead cells. The resulting single cell preparation was frozen in methanol (18) for subsequent single cell processing on the 10× Genomics platform, library construction and scRNA-Seq analysis. Some samples were processed fresh for comparison with methanol freezing.
Before proceeding with detailed analysis of many samples, it was sought to assess any bias that might be induced by methanol freezing of samples by the method of Chen (18). Thus, a comparison of the results from samples with single cell processing of fresh cells was made with processing after freezing in methanol. The results were nearly indistinguishable when comparing 10× genomics processing for scRNA-Seq using methanol-fixed compared to freshly processed ME cells for the scRNA-Seq analysis.
Having the established feasibility of this approach, an scRNA-Seq analysis of ME samples was made, from 11 subjects containing 3 controls, 4 patients with confirmed endometriosis, and 4 samples from patients with symptoms highly suggestive of endometriosis (and impaired decidualization). It was apparent from UMAP plots that multiple distinct clusters of cells can be delineated in these ME samples. This included stromal cells, NK cells, B cells, as well as several subsets of T cells and myeloid cells that cluster towards the center of the plot. A small number epithelial cells (<2-3% of total) were seen also. To understand the distribution of these various clusters comparing the control, endometriosis and symptomatic groups, the results were plotted separately for each group. It was apparent by simple inspection that the number of cells in some clusters are quite different between the clinical groups in particular, uNK cell, B cell, and some T cell subsets. An analysis of odds ratios (OR) for enrichment of these three clusters comparing endo and controls was performed. Both B cells and some T cell subsets are enriched by nearly 4-fold in the ME enriched for enzyme digested tissues of endo cases, while uterine NK (uNK) cells are very highly enriched in the ME of controls, compared with endometriosis cases. It is significant that in prior studies the B cells and T cells were not changed in endometriosis subjects versus control (see, e.g., US 2021-0096137 A1), yet, contrary to this, with the present methods a stark difference was seen in B cells and T cells were not changed in endometriosis subjects versus control.
Also examined were the patterns of gene expression in endometrial stromal cells between endometriosis cases and controls. Strikingly, IGFBP1 mRNA expression is markedly reduced in endometriosis patients compared with controls in the fresh cells. Thus, the data suggest that an analysis of IGFBP1 expression in fresh stromal cells in ME (Table 1) can in fact provide diagnostic power that is similar to the decidualization-induced IGFBP1 protein expression observed in cultured stromal cells following decidualization.
Table 1. Expression of IGFBP1 in fresh ME-derived stromal cells from endometriosis (ENDO) and control (Ctrl) subjects by scRNA-Seq.
|
% +
% +
|
Comparison
p_val
avg_log2FC
endo
control
p_val_adj
|
|
IGFBP1
1.55E−15
−1.79
0.2
0.4
3.35E−11
|
ENDO vs Ctrl
|
IGFBP1
4.77E−16
−1.62
0.2
0.4
1.03E−11
|
ENDO +
|
Sympto vs. Ctrl
|
|
In addition, scRNA-Seq permits study subsets of stromal cells and reveal additional gene expression differences between endometriosis patients (cases) and controls scRNA-Seq showed stromal cell subclusters display divergent gene expression patterns comparing controls and endometriosis cases. High expression of both IGFBP1 and LEFTY2 was seen in controls compared to endo in cluster 2, with fold changes in the range of 7-8 (log2˜2.6-2.8). These transcripts are strongly associated with decidualization. In contrast, cluster 1 was enriched for IL-11 and matrix metalloproteinases in cases with endometriosis.
Alternatively, qPCR may be performed as a transcript gene expression analysis. scRNA-Seq results show a striking increase in both IGFBP1 as well as LEFTY2, a late decidualization marker, in cluster 2 of ME-stromal cells from controls as compared with ME-stromal cells from endo cases. This again strongly suggests that the phenotype of reduced decidualization seen in cultured stromal cells can, surprisingly, in fact be captured in freshly isolated ME-derived endometrial stromal cells from endometriosis cases.
In view of these results, the inventors continued experimenting to find improved methods to enrich ME-endometrial tissues. The results of this scRNA seq analysis were compared to previous reports where free single cells in ME were collected and FACS analysis performed thereupon (14). In this recent approach, specific enrichment and selection for tissue fragments only was performed, filtering out all other free ME cells before tissue digestion and analysis. This is different from the process outlined above where tissue digestion of the entire contents of ME was done, thus including all the free cells in ME, (depleted of neutrophils and RBCs) as well as cells released from digested tissue. A comparison was made of scRNA-Seq of free cells in whole ME with the result after specific tissue fragment enrichment and collection on a 70 μm filter prior to digestion and scRNA-Seq. Analysis of scRNA-Seq results on undigested fresh ME (free single cells) compared with enriched/digested ME-endometrial tissues (right panels) was performed. One subject donated ME using a Diva Cup (Diva International, Inc., Kitchener, Ontario, Canada) (upper panels), the other utilized an external pad collection (lower panels). A dramatic enrichment of both stromal cells and epithelial cells was seen in samples derived from ME that is enriched for enzyme digested endometrial tissues.
A dramatic enrichment of both CD45neg stromal cells and epithelial cells was seen in the processed ME-endometrial tissue samples, compared with a fresh suspension of single cells in ME (without tissues). Interestingly, T cells, B cells and NK cell fractions are present in both types of samples. Of course, the subsets and gene expression patterns in these groups may be different between tissue and ME single cells, but in our view the results of most interest will be found in the cells derived from tissue.
PUBLICATIONS IN THE FIELD
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Patent Application
20250003981
